Methods for detection of a nucleic acid by sequential amplification

ABSTRACT

The invention relates to methods and compositions for detecting a target nucleic acid. The methods include subjecting a target, template, cleavage agent, polymerase and first, second and third oligonucleotide to reaction conditions that permit: (1) formation of a duplex between the target and first oligonucleotide; (2) extension of the first oligonucleotide; (3) cleavage of a first cleavage structure; (4) formation of a second duplex with a released flap; (5) extension of the released flap and (6) cleavage of a second cleavage structure within the third oligonucleotide. A signal generated by the release of the third oligonucleotide produces a signal that is detected and indicative of the presence of the target in the sample.

RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.10/981,942, filed Nov. 5, 2004, which is a divisional of applicationSer. No. 09/717,602, now U.S. Pat. No. 6,893,819, filed Nov. 21, 2000.The entire teachings of the above applications are incorporated hereinby reference.

BACKGROUND OF THE INVENTION

The fidelity of DNA replication, recombination, and repair is essentialfor maintaining genome stability, and all of these processes depend on5′ 3′ exonuclease enzymes which are present in all organisms. For DNArepair, these enzymes are required for damaged fragment excision andrecombinational mismatch correction. For replication, these nucleasesare critical for the efficient processing of Okazaki fragments duringlagging strand DNA synthesis. In Escherichia coli, this latter activityis provided by DNA polymerase I (PolI); E. Coli strains withinactivating mutations in the PolI 5′ 3′ exonuclease domain are notviable due to an inability to process Okazaki fragments. Eukaryotic DNApolymerases, however, lack an intrinsic 5′→3′ exonuclease domain, andthis critical activity is provided by the multifunctional,structure-specific metallonuclease FEN-1 (five′ exonuclease-1 or flapendonuclease-1), which also acts as an endonuclease for 5′ DNA flaps(Reviewed in Hosfield et al., 1998a, Cell, 95:135).

Methods of detecting and/or measuring a nucleic acid wherein an enzymeproduces a labeled nucleic acid fragment are known in the art.

U.S. Pat. Nos. 5,843,669, 5,719,028, 5,837,450, 5,846,717 and 5,888,780disclose a method of cleaving a target DNA molecule by incubating a 5′labeled target DNA with a DNA polymerase isolated from Thermus aquaticus(Taq polymerase) and a partially complementary oligonucleotide capableof hybridizing to sequences at the desired point of cleavage. Thepartially complementary oligonucleotide directs the Taq polymerase tothe target DNA through formation of a substrate structure containing aduplex with a 3′ extension opposite the desired site of cleavage whereinthe non-complementary region of the oligonucleotide provides a 3′ armand the unannealed 5′ region of the substrate molecule provides a 5′arm. The partially complementary oligonucleotide includes a 3′nucleotide extension capable of forming a short hairpin either whenunhybridized or when hybridized to a target sequence at the desiredpoint of cleavage. The release of labeled fragment is detected followingcleavage by Taq polymerase.

U.S. Pat. Nos. 5,843,669, 5,719,028, 5,837,450, 5,846,717 and 5,888,780disclose the generation of mutant, thermostable DNA polymerases thathave very little or no detectable synthetic activity, and wild typethermostable nuclease activity. The mutant polymerases are said to beuseful because they lack 5′ to 3′ synthetic activity; thus syntheticactivity is an undesirable side reaction in combination with a DNAcleavage step in a detection assay.

U.S. Pat. Nos. 5,843,669, 5,719,028, 5,837,450, 5,846,717 and 5,888,780disclose that wild type Taq polymerase or mutant Taq polymerases thatlack synthetic activity can release a labeled fragment by cleaving a 5′end labeled hairpin structure formed by heat denaturation followed bycooling, in the presence of a primer that binds to the 3′ arm of thehairpin structure. Further, U.S. Pat. Nos. 5,843,669, 5,719,028,5,837,450, 5,846,717 and 5,888,780 teach that the mutant Taq polymeraseslacking synthetic activity can also cleave this hairpin structure in theabsence of a primer that binds to the 3′ arm of the hairpin structure.

U.S. Pat. Nos. 5,843,669, 5,719,028, 5,837,450, 5,846,717 and 5,888,780also disclose that cleavage of this hairpin structure in the presence ofa primer that binds to the 3′ arm of the hairpin structure by mutant Taqpolymerases lacking synthetic activity yields a single species oflabeled cleaved product, while wild type Taq polymerase producesmultiple cleavage products and converts the hairpin structure to adouble stranded form in the presence of dNTPs, due to the high level ofsynthetic activity of the wild type Taq enzyme.

U.S. Pat. Nos. 5,843,669, 5,719,028, 5,837,450, 5,846,717 and 5,888,780also disclose that mutant Taq polymerases exhibiting reduced syntheticactivity, but not wild type Taq polymerase, can release a single labeledfragment by cleaving a linear nucleic acid substrate comprising a 5′ endlabeled target nucleic acid and a complementary oligonucleotide whereinthe complementary oligonucleotide hybridizes to a portion of the targetnucleic acid such that 5′ and 3′ regions of the target nucleic acid arenot annealed to the oligonucleotide and remain single stranded.

U.S. Pat. Nos. 5,846,717, 6,090,543, 6,001,567, 6,090,606, 5,985,557 and5,994,069 relate to methods for forming a nucleic acid cleavagestructure on a target sequence and cleaving the nucleic acid cleavagestructure in a site specific manner. These patents also relate to usingthe 5′ nuclease activity of a variety of enzymes to cleave thetarget-dependent cleavage structure, thereby indicating the presence ofa specific nucleic acid sequence or variations thereof.

Methods of detecting and/or measuring a nucleic acid wherein a FEN-1enzyme is used to generate a labeled nucleic acid fragment are known inthe art.

U.S. Pat. No. 5,843,669 discloses a method of detecting polymorphisms bycleavase fragment length polymorphism analysis using a thermostableFEN-1 nuclease in the presence or absence of a mutant Taq polymeraseexhibiting reduced synthetic activity. According to this method, doublestranded Hepatitis C virus (HCV) DNA fragments are labeled by using 5′end labeled primers (labeled with TMR fluorescent dye) in a PCRreaction. The TMR labeled PCR products are denatured by heating to 95°C. and cooled to 55° C. to generate a cleavage structure. U.S. Pat. No.5,843,669 discloses that a cleavage structure comprises a region of asingle stranded nucleic acid substrate containing secondary structure.Cleavage is carried out in the presence of CleavaseBN nuclease, FEN-1nuclease derived from the archaebacteria Methanococcus jannaschii orboth enzymes. Labeled reaction products are visualized by gelelectrophoresis followed by fluoroimaging. U.S. Pat. No. 5,843,669discloses that CleavaseBN nuclease and Methanococcus jannaschii FEN-1nuclease produce cleavage patterns that are easily distinguished fromeach other, and that the cleavage patterns from a reaction containingboth enzymes include elements of the patterns produced by cleavage witheach individual enzyme but are not merely a composite of the cleavagepatterns produced by each individual enzyme. This indicates that some ofthe fragments that are not cleaved by one enzyme (and which appear as aband in that enzyme's pattern) can be cleaved by a second enzyme in thesame reaction mixture.

Lyamichev et al. disclose a method for detecting DNAs whereinoverlapping pairs of oligonucleotide probes that are partiallycomplementary to a region of target DNA are mixed with the target DNA toform a 5′ flap region, and wherein cleavage of the labeled downstreamprobe by a thermostable FEN-1 nuclease produces a labeled cleavageproduct. Lyamichev et al. also disclose reaction conditions whereinmultiple copies of the downstream oligonucleotide probe can be cleavedfor a single target sequence in the absence of temperature cycling, soas to amplify the cleavage signal and allow quantitative detection oftarget DNA at sub-attomole levels (Lyamichev et al., 1999, Nat.Biotechnol., 17:292).

The polymerase chain reaction (PCR) technique, is disclosed in U.S. Pat.Nos. 4,683,202, 4,683,195 and 4,800,159. In its simplest form, PCR is anin vitro method for the enzymatic synthesis of specific DNA sequences,using two oligonucleotide primers that hybridize to opposite strands andflank the region of interest in the target DNA. A repetitive series ofreaction steps involving template denaturation, primer annealing and theextension of the annealed primers by DNA polymerase results in theexponential accumulation of a specific fragment whose termini aredefined by the 5′ ends of the primers. PCR is reported to be capable ofproducing a selective enrichment of a specific DNA sequence by a factorof 10⁹. The PCR method is also described in Saiki et al., 1985, Science,230:1350.

While the PCR technique is an extremely powerful method for amplifyingnucleic acid sequences, the detection of the amplified material requiresadditional manipulation and subsequent handling of the PCR products todetermine whether the target DNA is present. It is desirable to decreasethe number of subsequent handling steps currently required for thedetection of amplified material. An assay system, wherein a signal isgenerated while the target sequence is amplified, requires fewerhandling steps for the detection of amplified material, as compared to aPCR method that does not generate a signal during the amplificationstep.

U.S. Pat. Nos. 5,210,015 and 5,487,972 disclose a PCR based assay forreleasing labeled probe comprising generating a signal during theamplification step of a PCR reaction in the presence of a nucleic acidto be amplified, Taq polymerase that has 5′ to 3′ exonuclease activityand a 5′, 3′ or 5′ and 3′ end-labeled probe comprising a regioncomplementary to the amplified region and an additionalnon-complementary 5′ tail region. U.S. Pat. Nos. 5,210,015 and 5,487,972disclose further that this PCR based assay can liberate the 5′ labeledend of a hybridized probe when the Taq polymerase is positioned near thelabeled probe by an upstream probe in a polymerization independentmanner, e.g. in the absence of dNTPs.

There is a need in the art for a method of generating a signal that canbe easily distinguished from oligonucleotide fragments that may arisefrom nuclease contaminants, using a nucleic acid cleavage reaction.

There is also a need in the art for a method of generating a signalwherein a target is amplified in either a linear or an exponentialmanner.

There is a need in the art for a method of generating a signal wherein atarget is amplified in either a linear or an exponential manner in anisothermal reaction that utilizes a probe comprising secondary structurewherein some or all of the self-complementary regions of the probe thatanneal to form the secondary structure are melted when the probehybridizes with a target nucleic acid, thereby reducing non-specificbinding of the probe to the target, and increasing the specificity ofthe assay.

U.S. Pat. Nos. 5,843,669, 5,719,028, 5,837,450, 5,846,717 and 5,888,780also disclose a method of cleaving a labeled nucleic acid substrate atnaturally occurring areas of secondary structure. According to thismethod, biotin labeled DNA substrates are prepared by PCR, mixed withwild type Taq polymerase or CleavaseBN (a mutant Taq polymerase withreduced synthetic activity and wild type 5′ to 3′ nuclease activity),incubated at 95° C. for 5 seconds to denature the substrate and thenquickly cooled to 65° C. to allow the DNA to assume its unique secondarystructure by allowing the formation of intra-strand hydrogen bondsbetween the complementary bases. The reaction mixture is incubated at65° C. to allow cleavage to occur and biotinylated cleavage products aredetected.

There is a need in the art for a method of generating a signal using anucleic acid cleavage reaction wherein the cleavage structure is notrequired to contain areas of secondary structure and wherein a target isamplified in either a linear or an exponential manner.

SUMMARY OF THE INVENTION

The invention relates to nucleic acid, flap-mediated, sequentialamplification methods which permit detection of a nucleic acid target ina nucleic acid sample. The invention provides for nucleic acid synthesisdependent, flap-mediated amplification methods for sequentiallyproducing detectable, released flaps to detect a target nucleic acid.The invention provides for both linear, and exponential nucleic acidsynthesis dependent, flap-mediated, sequential amplification methods.The methods of the invention provide for either duplex structurescomprising a preformed flap or duplex structures wherein a flap isformed by the activity of a polymerization means with stranddisplacement activity. Nucleic acid synthesis dependent, flap-mediatedsequential amplification methods according to the invention comprise atemplate nucleic acid, a downstream oligonucleotide and an upstreamprimer. The invention may be performed isothermally or under nucleicacid amplifying conditions. The invention may be used in real-time PCRdetection.

In one embodiment, detectable released flaps are produced by a nucleicacid synthesis dependent, flap-mediated, sequential amplification methodwhich includes the step of providing one or more template nucleic acids(e.g., A′B′C′, FIG. 1, step 1), which comprise in 3′ to 5′ order a firstregion, an extension region, and a second region, an upstream primer(e.g., A, FIG. 1, step 1) that is at least partially complementary tothe first region of the template nucleic acid, and one or more copies ofa downstream oligonucleotide (e.g., AC, FIG. 1, step 1) comprising a 5′region and a 3′ region, wherein the 3′ region is at least partiallycomplementary to the second region of the template nucleic acid andwherein the 5′ region is at least partially complementary to a regionthat may be upstream of, downstream of, or comprise the first region ofthe template nucleic acid. One or more template nucleic acids refers toidentical or non-identical template nucleic acids. Non-identical means aleast one nucleotide (1, 2, 5, 10, 20 or more) is different.

This method also includes the step of forming a first duplex (FIG. 1,step 1) by mixing at least the template nucleic acid and the upstreamprimer and downstream oligonucleotide under conditions which permitformation of a duplex between the template nucleic acid and each of theupstream primer and the 3′ region of the downstream oligonucleotide,wherein the 5′ region of the downstream oligonucleotide is a flap. Afirst cleavage structure is then formed (FIG. 1, step 2) by subjectingthe duplex to a nucleic acid polymerization activity under conditionswhich permit extension of the upstream primer by polymerization of anucleic acid strand complementary to a length of the extension regionsufficient to form a first cleavage structure. The first cleavagestructure is cleaved (FIG. 1, step 2) by providing a cleavage meansunder conditions such that cleavage of the first cleavage structureoccurs at a site located within the downstream oligonucleotide in amanner dependent upon the formation of the first cleavage structure,thereby permitting cleavage and release of the flap of the downstreamoligonucleotide (e.g., A, FIG. 1, step 3).

The released flap of the downstream oligonucleotide associates witheither i) another copy of the template nucleic acid or ii) a denaturedversion of the template nucleic acid used to form the first duplex, andan uncleaved downstream oligonucleotide to form a second duplex (FIG. 1,step 4a or 4b) comprising the template nucleic acid and each of thereleased flap of the downstream oligonucleotide and an uncleaveddownstream oligonucleotide, wherein the 5′ region of the uncleaveddownstream oligonucleotide is a second flap. At this step, the releasedflap of the downstream oligonucleotide can associate with a templatenucleic acid that is identical (FIG. 1, step 4a) or non-identical (FIG.1, step 4b) to the template nucleic acid used to form the first duplex.

A second cleavage structure is formed (FIG. 1, step 5a or 5b) bysubjecting the second duplex to a nucleic acid polymerization activityunder conditions which permit extension of the released flap bypolymerization of a nucleic acid strand complementary to a length of theextension region sufficient to form a second cleavage structure. Asecond cleavage structure is cleaved (FIG. 1, step 5a or 5b) byproviding a cleavage means under conditions such that cleavage of thesecond cleavage structure occurs at a site located within the uncleaveddownstream oligonucleotide in a manner dependent upon the formation ofthe second cleavage structure, thereby permitting cleavage and releaseof the second flap (e.g., A, FIG. 1, step 6a, or A, FIG. 1, step 6b).Released first and/or second flaps are detected. These steps can berepeated, for example 1-2 million times and preferably 1-10,000 times,to produce a detectable level of first and/or second flaps.

The invention also provides for linear synthesis dependent,flap-mediated, sequential amplification methods for target nucleic aciddetection.

In one embodiment, duplex structures comprise a preformed flap. Thisembodiment of the invention comprises the step of providing thefollowing: a target nucleic acid (e.g., A′B′C′, FIG. 2, step 1), whichcomprises in 3′ to 5′ order a first region, an extension region, and asecond region; one or more template nucleic acids (e.g., F′G′H′, FIG. 2,step 4), which comprise in 3′ to 5′ order a first region, an extensionregion, and a second region; a first oligonucleotide (e.g., A, FIG. 1,step 1) that is at least partially complementary to the first region ofthe target nucleic acid; a second oligonucleotide (e.g., FC, FIG. 2,step 1) comprising a 5′ region and a 3′ region, wherein the 3′ region isat least partially complementary to the second region of the targetnucleic acid and wherein the 5′ region is not complementary to theextension region of the target nucleic acid but is at least partiallycomplementary to the first region of the template nucleic acid; and oneor more third oligonucleotides comprising a 5′ region and a 3′ region,wherein the 3′ region is at least partially complementary to the secondregion of the template nucleic acid and the 5′ region is notcomplementary to the template nucleic acid.

According to this embodiment, a first duplex is formed (FIG. 2, step 1)by mixing at least the target nucleic acid and the first and secondoligonucleotides under conditions which permit formation of a duplexbetween the target nucleic acid and each of the first oligonucleotideand the 3′ region of the second oligonucleotide, wherein the 5′ regionof the second oligonucleotide is a flap. A first cleavage structure isformed (FIG. 2, step 2) by subjecting the duplex to a nucleic acidpolymerization activity under conditions which permit extension of thefirst oligonucleotide by polymerization of a nucleic acid strandcomplementary to a length of the extension region sufficient to form afirst cleavage structure. A first cleavage structure is cleaved (FIG. 2,step 2) by providing a cleavage means under conditions such thatcleavage of the first cleavage structure occurs at a site located withinthe second oligonucleotide in a manner dependent upon the formation ofthe first cleavage structure, thereby permitting cleavage and release ofthe flap (e.g., F, FIG. 2, step 3) of the second oligonucleotide. Thereleased flap of the second oligonucleotide, the template nucleic acid,and the third oligonucleotide form a second duplex (FIG. 2, step 4)between the template nucleic acid and each of the released flap of thesecond oligonucleotide and the third oligonucleotide, wherein the 5′region of the third oligonucleotide is a second flap. A second cleavagestructure is formed (FIG. 2, step 5) by subjecting the second duplex toa nucleic acid polymerization activity under conditions which permitextension of the released flap by polymerization of a nucleic acidstrand complementary to a length of the extension region sufficient toform a second cleavage structure. A second cleavage structure is cleaved(FIG. 2, step 5) by providing a cleavage means under conditions suchthat cleavage of the second cleavage structure occurs at a site locatedwithin the third oligonucleotide in a manner dependent upon theformation of the second cleavage structure, thereby permitting cleavageand release of the second flap of the third oligonucleotide (e.g., F,FIG. 2, step 6). Released first and/or second flaps are detected. Thesesteps can be repeated, for example 1-2 million times, preferably1-10,000 times, to produce a detectable level of first and second flaps.

In a related embodiment, a linear nucleic acid synthesis dependent,flap-mediated, sequential amplification method for detecting a targetnucleic acid comprises cleavage structures wherein the flap of at leastone of the cleavage structures is formed by the activity of apolymerization means with strand displacement activity (see FIG. 3).According to this embodiment, at least 6 nucleotides are displaced bythe strand displacement activity of the polymerization means (FIG. 3,step 2). A second oligonucleotide according to this embodiment, furthercomprises a 5′ region that is at least partially complementary to aregion that may be upstream of, downstream of, or comprising the firstregion of the target nucleic acid (e.g., FC, FIG. 3, step 1). A thirdoligonucleotide according to this embodiment of the invention furthercomprises a 5′ region that is at least partially complementary to aregion that may be upstream of, downstream of, or comprising the firstregion of the template nucleic acid (e.g., FH, FIG. 3, step 4).

The invention provides for compositions for linear nucleic acidsynthesis dependent, flap-mediated amplification methods forsequentially producing detectable, released flaps to detect a targetnucleic acid.

The invention provides for a composition comprising one or more templatenucleic acids, each of which comprises in 3′ to 5′ order a first region,an extension region, and a second region; a first oligonucleotide thatis at least partially complementary to the first region of the templatenucleic acid, and one or more second oligonucleotides each comprising a5′ region and a 3′ region, wherein the 3′ region of each secondoligonucleotide is at least partially complementary to the second regionof the template nucleic acid and wherein the 5′ region is at leastpartially complementary to a corresponding region that may be upstreamof, downstream of, or comprise the first region of the template nucleicacid.

A composition comprising: a target nucleic acid, which comprises in 3′to 5′ order a first region, an extension region, and a second region, atemplate nucleic acid, which comprises in 3′ to 5′ order a first region,an extension region, and a second region, a first oligonucleotide thatis at least partially complementary to the first region of the targetnucleic acid, and a second oligonucleotide comprising a 5′ region and a3′ region, wherein the 3′ region is at least partially complementary tothe second region of the target nucleic acid and wherein the 5′ regionis not complementary to the extension region of the target nucleic acidbut is at least partially complementary to the first region of thetemplate nucleic acid; and a third oligonucleotide comprising a 5′region and a 3′ region, wherein the 3′ region is at least partiallycomplementary to the second region of the template nucleic acid and the5′ region is not complementary to the first region of the templatenucleic acid.

A composition comprising: a target nucleic acid, which comprises in 3′to 5′ order a first region, an extension region, and a second region, atemplate nucleic acid, which comprises in 3′ to 5′ order a first region,an extension region, and a second region, a first oligonucleotide thatis at least partially complementary to the first region of the targetnucleic acid, and a second oligonucleotide comprising a 5′ region and a3′ region, wherein the 3′ region is at least partially complementary tothe second region of the target nucleic acid and wherein the 5′ regionis at least partially complementary to a region that may be upstream of,downstream of, or comprising the first region of the target nucleic acidand is at least partially complementary to the first region of thetemplate nucleic acid; and a third oligonucleotide comprising a 5′region and a 3′ region, wherein the 3′ region is at least partiallycomplementary to the second region of the template nucleic acid and the5′ region is at least partially complementary to a region that may beupstream of, downstream of, or comprising the first region of thetemplate nucleic acid.

Preferably, in any one of the compositions, the composition may furtherinclude either or both of a cleavage means which permits cleavage of aflap from a nucleic acid duplex structure, and a nucleic acidpolymerizing activity.

A kit comprising any one of the compositions including a target nucleicacid as described hereinabove, and packaging materials therefore.

Preferably, the kit may further include either or both of a cleavagemeans which permits cleavage of a flap from a nucleic acid duplexstructure, and a nucleic acid polymerizing activity.

The invention contemplates both linear and exponential amplificationmethods. The invention provides for exponential, synthesis dependent,flap-mediated, amplification methods for sequentially producing releasedflaps for detecting a target nucleic acid.

In one embodiment, detectable released flaps are produced by a nucleicacid synthesis dependent, flap-mediated, sequential amplification methodwhich includes the step of providing: one or more template nucleic acids(e.g., F′G1′H1′G2′H2′, FIG. 4A, step 1), which comprise in 3′ to 5′order a first region, a first extension region, a second region, asecond extension region, and a third region, an upstream primer (e.g.,F, FIG. 4 a, step 1) that is at least partially complementary to thefirst region of the template nucleic acid, and one or more copies of afirst downstream oligonucleotide (e.g., FH1, FIG. 4 a, step 1)comprising a 5′ region and a 3′ region, wherein the 3′ region is atleast partially complementary to the second region of the templatenucleic acid and wherein the 5′ region is at least partiallycomplementary to a region that may be upstream of, downstream of, orcomprise the first region of the template nucleic acid; and a seconddownstream oligonucleotide (e.g., FH2, FIG. 4 a, step 1) which isdownstream of the first downstream oligonucleotide and which comprises a5′ region and a 3′ region, wherein the 3′ region is at least partiallycomplementary to the third region of the template nucleic acid andwherein the 3′ region of the first downstream oligonucleotide may or maynot be identical in sequence to the 3′ region of the second downstreamoligonucleotide, and wherein the 5′ region of the second downstreamoligonucleotide is at least partially complementary to a region that maybe upstream of, downstream of, or comprise the first region of thetemplate nucleic acid. One or more template nucleic acids refers toidentical or non-identical template nucleic acids.

This method also includes the step of forming a first duplex (FIG. 4 a,step 1) by mixing at least the template nucleic acid and the upstreamprimer and the first and second downstream oligonucleotides underconditions which permit formation of a duplex between the templatenucleic acid and each of the upstream primer and the 3′ regions of thefirst and second downstream oligonucleotides, wherein the 5′ regions ofthe first and second downstream oligonucleotides are flaps.

A first cleavage structure is then formed (FIG. 4 a, step 2) bysubjecting the duplex to a nucleic acid polymerization activity underconditions which permit extension of the upstream primer bypolymerization of a nucleic acid strand complementary to a length of thefirst extension region sufficient to form a first cleavage structure.The first cleavage structure is cleaved (FIG. 4 a, step 2) by providinga cleavage means under conditions such that cleavage of the firstcleavage structure occurs at a site located within the first downstreamoligonucleotide in a manner dependent upon the formation of the firstcleavage structure, thereby permitting cleavage and release of the flapof the first downstream oligonucleotide (e.g., F, FIG. 4 a, step 3).

A second cleavage structure is then formed (FIG. 4 a, steps 3 and 4) bysubjecting the duplex to a nucleic acid polymerization activity underconditions which permit extension of the upstream primer bypolymerization of a nucleic acid strand complementary to a length of thesecond extension region sufficient to form a second cleavage structure.According to this embodiment, the polymerization activity causesdisplacement of the uncleaved portion of the first downstreamoligonucleotide (e.g., H1, FIG. 4 a, step 3).

The second cleavage structure is cleaved (FIG. 4 a, step 4) by providinga cleavage means under conditions such that cleavage of the secondcleavage structure occurs at a site located within the second downstreamoligonucleotide in a manner dependent upon the formation of the secondcleavage structure, thereby permitting cleavage and release of the flapof the second downstream oligonucleotide (e.g., F, FIG. 4 a, step 5).The released flap of the first or second downstream oligonucleotideassociates with either i) another copy of the template nucleic acid orii) a denatured version of the template nucleic acid used to form thefirst duplex, and uncleaved first and second downstream oligonucleotidesto form a second duplex (FIG. 4 a, step 1 or FIG. 4 b, step 6)comprising the template nucleic acid and either of the released flap ofthe first or second downstream oligonucleotide, uncleaved firstdownstream oligonucleotide, and uncleaved second downstreamoligonucleotide, wherein the 5′ region of each uncleaved downstreamoligonucleotide, independently, is a flap. At this step, the releasedflap of the first or second downstream oligonucleotide can associatewith a template nucleic acid that is identical (e.g., F′G1′H1′G2′H2′,FIG. 4 a, step 1) or non-identical (e.g., F′I1′H1′I2′H2′, FIG. 4 b, step6) to the template nucleic acid used to form the first duplex.

A third cleavage structure is formed (FIG. 4 a, step 2 or FIG. 4 b, step7) by subjecting the second duplex to a nucleic acid polymerizationactivity under conditions which permit extension of the released flap bypolymerization of a nucleic acid strand complementary to a length of thefirst extension region sufficient to form a third cleavage structure. Athird cleavage structure is cleaved (FIG. 4 a, step 2 or FIG. 4 b, step7) by providing a cleavage means under conditions such that cleavage ofthe third cleavage structure occurs at a site located within theuncleaved first downstream oligonucleotide in a manner dependent uponthe formation of the third cleavage structure, thereby permittingcleavage and release of the third flap (e.g., F, FIG. 4 a, step 3 or F,FIG. 4 b, step 8).

A fourth cleavage structure is then formed (FIG. 4 a, steps 3 and 4 orFIG. 4 b, steps 8 and 9) by subjecting the duplex to a nucleic acidpolymerization activity under conditions which permit extension of theupstream primer by polymerization of a nucleic acid strand complementaryto a length of the second extension region sufficient to form a fourthcleavage structure. According to this embodiment, the polymerizationactivity causes displacement of the uncleaved portion of the firstdownstream oligonucleotide.

The fourth cleavage structure is cleaved (FIG. 4 a, step 4 or FIG. 4 b,step 9) by providing a cleavage means under conditions such thatcleavage of the fourth cleavage structure occurs at a site locatedwithin the second downstream oligonucleotide in a manner dependent uponthe formation of the fourth cleavage structure, thereby permittingcleavage and release of the flap of the second downstreamoligonucleotide (e.g., F, FIG. 4 a, step 5 or F, FIG. 4 b, step 10).Released first and/or second and/or third and/or fourth flaps aredetected. These steps can be repeated, for example 1-100 times andpreferably 1-50 times, to produce a detectable level of first and/orsecond flaps.

In one embodiment, duplex structures comprise a preformed flap. Thisembodiment of the invention comprises the step of providing: a targetnucleic acid (e.g., A′B′C′, FIG. 2, step 1) suspected of containing thenucleic acid of interest, which comprises in 3′ to 5′ order a firstregion, an extension region, and a second region; a template nucleicacid (e.g., F′G1′H1′G2′H2′, FIG. 5, step 1), which comprises in 3′ to 5′order a first region, a first extension region, a second region, asecond extension region and a third region; a first oligonucleotide(e.g., A, FIG. 2, step 1) that is at least partially complementary tothe first region of the target nucleic acid; a second oligonucleotide(e.g., FC, FIG. 2, step 1) comprising a 5′ region and a 3′ region,wherein the 3′ region is at least partially complementary to the secondregion of the target nucleic acid and wherein the 5′ region is notcomplementary to the extension region of the target nucleic acid but isat least partially complementary to the first region of the templatenucleic acid; a third oligonucleotide (e.g., FH1, FIG. 5, step 1)comprising a 5′ region and a 3′ region, wherein the 3′ region is atleast partially complementary to the second region of the templatenucleic acid, and wherein the 5′ region is not complementary to thefirst extension region of the template nucleic acid, and a fourtholigonucleotide (e.g., FH2, step 1) comprising a 5′ region and a 3′region, wherein the 3′ region is at least partially complementary to thethird region of the template nucleic acid, and wherein the 3′ region ofthe third oligonucleotide may or may not be identical in sequence to the3′ region of the fourth oligonucleotide, and wherein the 5′ region ofthe fourth oligonucleotide is not complementary to the second extensionregion of the template nucleic acid.

This method also includes the step of forming a first duplex (FIG. 2,step 1) by mixing at least the target nucleic acid and the first andsecond oligonucleotides under conditions which permit formation of aduplex between the target nucleic acid and each of the firstoligonucleotide and the 3′ region of the second oligonucleotide, whereinthe 5′ region of the second oligonucleotide is a first flap. A firstcleavage structure is formed (FIG. 2, step 2) by subjecting the duplexto a nucleic acid polymerization activity under conditions which permitextension of the first oligonucleotide by polymerization of a nucleicacid strand complementary to a length of the extension region sufficientto form a first cleavage structure. A first cleavage structure iscleaved (FIG. 2, step 2) by providing a cleavage means under conditionssuch that cleavage of the first cleavage structure occurs in the secondoligonucleotide in a manner dependent upon the formation of the firstcleavage structure, thereby permitting cleavage and release of the firstflap of the second oligonucleotide (e.g., F, FIG. 2, step 3). Thereleased first flap of the second oligonucleotide (e.g., F, FIG. 2, step3), the template nucleic acid, the third oligonucleotide, and the fourtholigonucleotide associate to form a second duplex (FIG. 4, step 1),wherein the 5′ region of the third oligonucleotide is a second flap, andthe 5′ region of the fourth oligonucleotide is a third flap.

A second cleavage structure is formed (FIG. 5, step 1) by subjecting thesecond duplex to a nucleic acid polymerization activity under conditionswhich permit extension of the released first flap of the second duplexby polymerization of a nucleic acid strand complementary to a length ofthe first extension region of the second duplex sufficient to form asecond cleavage structure.

A second cleavage structure is cleaved (FIG. 5, step 2) by providing acleavage means under conditions such that cleavage of the secondcleavage structure occurs in the third oligonucleotide in a mannerdependent upon the formation of the second cleavage structure, therebypermitting cleavage and release of the second flap of the thirdoligonucleotide (e.g., F, FIG. 5, step 3) and cleavage of the secondcleavage structure.

A third cleavage structure is formed (FIG. 5, steps 3 and 4) bysubjecting the second duplex to a nucleic acid polymerization activityunder conditions which permit extension of the released first flap bypolymerization of a nucleic acid strand complementary to a length of thesecond extension region of the cleaved second cleavage structuresufficient to form a third cleavage structure. According to thisembodiment, the polymerization activity causes displacement of theuncleaved portion of the third oligonucleotide (e.g., H1, FIG. 5, step3). A third cleavage structure is cleaved (FIG. 5, step 4) by providinga cleavage means under conditions such that cleavage of the thirdcleavage structure occurs in the fourth oligonucleotide in a mannerdependent upon the formation of the third cleavage structure, therebypermitting cleavage and release of the third flap of the fourtholigonucleotide (e.g., F, FIG. 5, step 5). At least one of the first,second or third released flaps are detected.

In a related embodiment, an exponential, synthesis dependent,flap-mediated, sequential amplification method for detecting a targetnucleic acid comprises first, second and third cleavage structureswherein the flap of at least one of the first, second and third cleavagestructures is formed by the activity of a polymerization means withstrand displacement activity (see FIG. 6). According to this embodiment,a polymerization means displaces at least 6 nucleotides. Duplexstructures according to this embodiment, do not comprise a preformedflap.

A second oligonucleotide according to this embodiment further comprisesa 5′ region that is at least partially complementary to a region thatmay be upstream of, downstream of, or comprising the first region of thetarget nucleic acid. A third oligonucleotide according to thisembodiment, further comprises a 5′ region that is at least partiallycomplementary to a region that may be upstream of, downstream of orcomprising the first region of the template nucleic acid. A fourtholigonucleotide according to this embodiment, further comprises a 5′region that is at least partially complementary to a region that may beupstream of, downstream of, or comprising the second region of thetemplate nucleic acid.

The invention provides for compositions for exponential nucleic acidsynthesis dependent, flap-mediated amplification methods forsequentially producing detectable, released flaps to detect a targetnucleic acid.

The invention provides for a composition comprising one or more templatenucleic acids, which comprise in 3′ to 5′ order a first region, a firstextension region, a second region, a second extension region, and athird region, an upstream primer that is at least partiallycomplementary to the first region of the template nucleic acid; one ormore copies of a first downstream oligonucleotide comprising a 5′ regionand a 3′ region, wherein the 3′ region is at least partiallycomplementary to the second region of the template nucleic acid andwherein the 5′ region is at least partially complementary to a regionthat may be upstream of, downstream of, or comprise the first region ofthe template nucleic acid; one or more copies of a second downstreamoligonucleotide comprising a 5′ region and a 3′ region, wherein the 3′region is at least partially complementary to the third region of thetemplate nucleic acid and wherein the 5′ region is at least partiallycomplementary to a region that may be upstream of, downstream of, orcomprise the second region of the template nucleic acid.

A composition comprising: a target nucleic acid suspected of containingthe nucleic acid of interest, which comprises in 3′ to 5′ order a firstregion, an extension region, and a second region, a template nucleicacid, which comprises in 3′ to 5′ order a first region, a firstextension region, a second region, a second extension region and a thirdregion, a first oligonucleotide that is at least partially complementaryto the first region of the target nucleic acid, and a secondoligonucleotide comprising a 5′ region and a 3′ region, wherein the 3′region is at least partially complementary to the second region of thetarget nucleic acid and wherein the 5′ region is not complementary tothe extension region of the target nucleic acid but is at leastpartially complementary to the first region of the template nucleicacid; and a third oligonucleotide comprising a 5′ region and a 3′region, wherein the 3′ region is at least partially complementary to thesecond region of the template nucleic acid, and wherein the 5′ region isnot complementary to the first extension region of the template nucleicacid, and a fourth oligonucleotide comprising a 5′ region and a 3′region, wherein the 3′ region is at least partially complementary to thethird region of the template nucleic acid, and wherein the 3′ region ofthe third oligonucleotide is not identical in sequence to the 3′ regionof the fourth oligonucleotide, and wherein the 5′ region of the fourtholigonucleotide is not complementary to the second extension region ofthe template nucleic acid.

A composition comprising: a target nucleic acid suspected of containingthe nucleic acid of interest, which comprises in 3′ to 5′ order a firstregion, an extension region, and a second region, a template nucleicacid, which comprises in 3′ to 5′ order a first region, a firstextension region, a second region, a second extension region and a thirdregion, a first oligonucleotide that is at least partially complementaryto the first region of the target nucleic acid, and a secondoligonucleotide comprising a 5′ region and a 3′ region, wherein the 3′region is at least partially complementary to the second region of thetarget nucleic acid and wherein the 5′ region is at least partiallycomplementary to a region that may be upstream of, downstream of, orcomprising the first region of the target nucleic acid and is at leastpartially complementary to the first region of the template nucleicacid; and a third oligonucleotide comprising a 5′ region and a 3′region, wherein the 3′ region is at least partially complementary to thesecond region of the template nucleic acid, and wherein the 5′ region isat least partially complementary to a region that may be upstream of,downstream of, or comprising the first region of the template nucleicacid, and a fourth oligonucleotide comprising a 5′ region and a 3′region, wherein the 3′ region is at least partially complementary to thethird region of the template nucleic acid, and wherein the 3′ region ofthe third oligonucleotide is not identical in sequence to the 3′ regionof the fourth oligonucleotide, and wherein the 5′ region of the fourtholigonucleotide is at least partially complementary to a region that maybe upstream of, downstream of, or comprising the second region of thetemplate nucleic acid.

Preferably, in any one of the compositions, the composition may furtherinclude either or both of a cleavage means which permits cleavage of aflap from a nucleic acid duplex structure, and a nucleic acidpolymerizing activity.

A kit comprising any one of the compositions including a target nucleicacid as described hereinabove, and packaging materials therefore.

Preferably, the kit may further include either or both of a cleavagemeans which permits cleavage of a flap from a nucleic acid duplexstructure, and a nucleic acid polymerizing activity.

In another aspect, the invention provides for a method of detecting atarget nucleic acid by: providing a set of reagents, mixing the set ofreagents, and detecting a signal (See FIG. 17). The method of theinvention includes providing a target nucleic acid (FIG. 17; A′B′C′), atemplate nucleic acid (FIG. 17; H′G′F′), a first oligonucleotide (FIG.17; A), a second oligonucleotide (FIG. 17; FC), a third oligonucleotide(FIG. 17; H), a cleavage means and a polymerization activity. The targetand template nucleic acids have, in 3′ to 5′ order, a first region, anextension region, and a second region. The first oligonucleotide is atleast partially complementary to the first region of the target nucleicacid. The second oligonucleotide's 3′ region is at least partiallycomplementary to the second region of the target nucleic acid and its 5′region is not complementary to the target nucleic acid but is at leastpartially complementary to the first region of the template nucleicacid. The third oligonucleotide's 3′ region is at least partiallycomplementary to the second region of the template nucleic acid and its5′ region is not complementary to the template nucleic acid.

The reagents are mixed under a series of reaction conditions thatpermit: formation of a duplex between the target and the first andsecond oligonucleotides, extension of the first oligonucleotide,cleavage of a first cleavage structure, formation of a second duplex,extension of a released flap, and formation of a second cleavagestructure. The target nucleic acid, template nucleic acid, firstoligonucleotide, second oligonucleotide, third oligonucleotide, cleavagemeans and polymerization activity are mixed under the series of reactionconditions. The reaction conditions permit formation of a duplex betweenthe target nucleic acid and each of the first oligonucleotide and the 3′region of the second oligonucleotide (FIG. 17A). The 5′ region of thesecond oligonucleotide forms a flap. The first oligonucleotide isextended by polymerization of a nucleic acid strand complementary to alength of the extension region of the target nucleic acid sufficient toform a first cleavage structure (FIG. 17B). The first cleavage structureis then cleaved within the second oligonucleotide by the cleavage means,thereby permitting release of the flap of the second oligonucleotide(FIG. 17C). A second duplex is formed with the released flap of thesecond oligonucleotide, the template nucleic acid, and the thirdoligonucleotide (FIG. 17D). The 5′ region of the third oligonucleotideforms a second flap. The released flap is then extended bypolymerization of a nucleic acid strand complementary to a length of theextension region of the template nucleic acid sufficient to form asecond cleavage structure (FIG. 17E). The second cleavage structure isthen cleaved within the third oligonucleotide by the cleavage means,thereby permitting release of the third oligonucleotide (FIG. 17F). Thesignal generated by the release of the third oligonucleotide from thetemplate nucleic acid is detected.

In yet another aspect, the invention provides for a method of detectinga target nucleic acid by: providing a set of reagents, mixing the set ofreagents, and detecting a signal. The method includes providing a targetnucleic acid, a template nucleic acid, a first oligonucleotide, a secondoligonucleotide, a third oligonucleotide, a cleavage means, and apolymerization activity. The target and template nucleic acids have, in3′ to 5′ order, a first region, an extension region, and a secondregion. The first oligonucleotide is at least partially complementary tothe first region of the target nucleic acid. The second oligonucleotidehas a 3′ region that is at least partially complementary to the secondregion of the target nucleic acid and a 5′ region that is notcomplementary to the target nucleic acid but is at least partiallycomplementary to the first region of the template nucleic acid. Thethird oligonucleotide includes a 3′ region that is at least partiallycomplementary to the second region of the template nucleic acid and a 5′region that is at least partially complementary to a region that is 3′of the second region.

The reagents are mixed under a series of reaction conditions that permitformation of a duplex between the target and the first and secondoligonucleotides, extension of the first oligonucleotide, cleavage of afirst cleavage structure, formation of a second duplex, extension of areleased flap, and formation of a second cleavage structure. The targetnucleic acid, template nucleic acid, first oligonucleotide, secondoligonucleotide, third oligonucleotide, cleavage means andpolymerization activity are mixed under the series of reactionconditions. The reaction conditions permit formation of a duplex betweenthe target nucleic acid and each of the first oligonucleotide and the 3′region of the second oligonucleotide. The 5′ region of the secondoligonucleotide forms a flap. The first oligonucleotide is extended bypolymerization of a nucleic acid strand complementary to a length of theextension region of the target nucleic acid sufficient to form a firstcleavage structure. The first cleavage structure, within the secondoligonucleotide, is cleaved by the cleavage means, thereby permittingrelease of the flap of the second oligonucleotide. A second duplex isformed with the released flap of the second oligonucleotide, thetemplate nucleic acid, and the third oligonucleotide. The released flapis extended by polymerization of a nucleic acid strand complementary toa length of the extension region of the template nucleic acid sufficientto form a second cleavage structure. The second cleavage structure,within the third oligonucleotide, is then cleaved by the cleavage means,thereby permitting release of the third oligonucleotide. A signalgenerated by the release of the third oligonucleotide from the templatenucleic acid is detected.

In either of the last two aspects of the invention, the polymerizationactivity and cleavage means may be present in a single enzyme. Suchenzymes providing both activities include E. coli DNA polymerase I, T7DNA polymerase, Tth DNA polymerase, or Taq DNA polymerase. In analternative embodiment, the polymerization activity and cleavage meansare each provided by a separate enzyme, e.g., a Pfu DNA polymerase and aFEN-1 nuclease. The polymerase activity is provided by a polymerase thatmay be a DNA polymerase and/or thermostable. The polymerase may lack a5′ to 3′ exonuclease activity. The cleavage means can include a 5′nuclease activity. Such 5′ nuclease activity is present in a FEN-1nuclease. The FEN-1 nuclease may be a flap specific nuclease. In oneembodiment, the FEN-1 nuclease is thermostable.

In yet another embodiment of either of the last two aspects of theinvention, the series of reaction conditions are nucleic acidpolymerization reaction conditions. In a further embodiment, the nucleicacid polymerization reaction conditions are polymerase chain reactionconditions.

In some embodiment, the cleavage structures are formed when thepolymerized complementary nucleic acid is adjacent at its 3′ end to thedownstream oligonucleotide in the duplex. In another embodiment, thecleavage structures are formed when the extension regions are fullyduplexed. In yet another embodiment, the cleavage structures are formedwhen the template strand of the extension region has a two, four, six,eight or ten nucleotide gap between the 3′ end of the complementarypolymerized nucleic acid and the downstream oligonucleotide.

In some embodiments of either of the last two aspects of the invention,the template nucleic acid is operably coupled to a first member of apair of interactive labels and the third oligonucleotide is operablycoupled to a second member of the pair of interactive labels. The firstmember may be operatively coupled to the 5′ nucleotide of the templatenucleic acid and the second member may be operatively coupled to the 3′nucleotide of the third oligonucleotide. In one embodiment, the pair ofinteractive levels is a fluorophore and a quencher. In the method of theinvention, the signal may be detected by determining a change influorescence between the first and second members of the pair ofinteractive labels upon release of the third oligonucleotide.

In some embodiments of the invention, the second oligonucleotide is notdetectably labeled. In yet another embodiment of the invention, the 5′portion of the second oligonucleotide is at least partiallycomplementary to the 3′ portion of the second oligonucleotide. In yet afurther embodiment, the 3′ portion and the 5′ portion of the secondoligonucleotide form a stem structure when the second oligonucleotide isnot hybridize to the target nucleic acid. The second oligonucleotide mayinclude a linker sequence between the 5′ portion and the 3′ portion ofthe second oligonucleotide. The linker portion may not be complementaryto the target nucleic acid but can be complementary to the first portionof the template nucleic acid. The 3′ nucleotide of the secondoligonucleotide and/or the template nucleic acid may be blocked.

In yet another aspect of the invention, the invention is directed to acomposition including a target nucleic acid, a template nucleic acid, afirst oligonucleotide, a second oligonucleotide and a thirdoligonucleotide. The target and template nucleic acids have, in 3′ to 5′order, a first region, an extension region, and a second region. Thetemplate nucleic acid has a first member of a pair of interactivelabels. The first oligonucleotide is at least partially complementary tothe first region of the target nucleic acid. The secondoligonucleotide's 3′ region is at least partially complementary to thesecond region of the target nucleic acid and its 5′ region is notcomplementary to the target nucleic acid but is at least partiallycomplementary to the first region of the template nucleic acid. Thethird oligonucleotide's 3′ region is at least partially complementary tothe second region of the template nucleic acid, while its 5′ region isnot complementary to the template nucleic acid. The thirdoligonucleotide is operatively coupled to a second member of the pair ofinteractive labels.

In yet another aspect, the invention is directed to a composition havinga target nucleic acid, template nucleic acid, first oligonucleotide,second oligonucleotide and a third oligonucleotide. The target andtemplate nucleic acids include, in 3′ to 5′ order, a first region, anextension region, and a second region. The template nucleic acid isoperable coupled to a first member of a pair of interactive labels. Thefirst oligonucleotide is at least partially complementary to the firstregion of the target nucleic acid. The second oligonucleotide's 3′region is at least partially complementary to the second region of thetarget nucleic acid and its 5′ region is not complementary to the targetnucleic acid but is at least partially complementary to the first regionof the template nucleic acid. The third oligonucleotide's 3′ region isat least partially complementary to the second region of the templatenucleic acid and its 5′ region is at least partially complementary to aregion that is 3′ of the second region of the template nucleic acid. Thethird oligonucleotide is operatively coupled to a second member of thepair of interactive labels.

In one embodiment of either of the last two aspects, the compositionsfurther include a cleavage means. In yet a further embodiment, thecompositions also include a polymerase. The polymerase and cleavagemeans may be present in a single enzyme. Such enzymes providing bothactivities include E. coli DNA polymerase I, T7 DNA polymerase, Tth DNApolymerase, or Taq DNA polymerase. In an alternative embodiment, thepolymerization activity and cleavage means are each provided by aseparate enzyme, e.g., a Pfu DNA polymerase and a FEN-1 nuclease. Thepolymerase activity is provided by a polymerase that may be a DNApolymerase and/or thermostable. The polymerase may lack a 5′ to 3′exonuclease activity. The cleavage means can include a 5′ nucleaseactivity. Such 5′ nuclease activity is present in a FEN-1 nuclease. TheFEN-1 nuclease may be a flap specific nuclease. The cleavage means maybe a FEN-1 nuclease and the polymerase may be a Pfu DNA Polymerase.

In another embodiment of the compositions, the 3′ nucleotide of thesecond oligonucleotide is blocked and/or the 3′ nucleotide of thetemplate nucleic acid is blocked. In some embodiments, the secondoligonucleotide may not have a detectable label.

In some embodiments of either of the last two aspects of the invention,the pair of interactive labels includes a fluorophore and a quencher. Inyet another embodiment, the second member of the pair of interactivelabels is operatively coupled to the 3′ nucleotide of the thirdoligonucleotide and/or the first member of the pair of interactivelabels is operatively coupled to the 5′ nucleotide of the templatenucleic acid.

In yet another embodiment, the 5′ portion of the second oligonucleotideis at least partially complementary to the 3′ portion of the secondoligonucleotide. In a further embodiment, the 3′ portion and the 5′portion of the second oligonucleotide form a stem structure when thesecond oligonucleotide is not hybridize to the target nucleic acid. Thesecond oligonucleotide may include a linker sequence between the 5′portion and the 3′ portion of the second oligonucleotide. In someembodiments, the linker sequence is not complementary to the targetnucleic acid but is complementary to the first portion of the templatenucleic acid.

A kit comprising any one of the compositions as described hereinabove,and packaging materials therefore.

As used herein, a “target nucleic acid” refers to a polynucleotide whichcomprises in 3′ to 5′ order a first region that is complementary to atleast a portion of a first oligonucleotide, an extension region and asecond region that complementary to at least a portion of a secondoligonucleotide. The target nucleic acid may comprise single ordouble-stranded DNA or RNA.

As used herein, a “first region” as it refers to a target nucleic acid,means a length of nucleotides sufficient to permit hybridization andextension of a first oligonucleotide wherein the “first region” iscomplementary to at least a portion of a first oligonucleotide, definedherein. A “first region” is in the range of about 6 nucleotides to about1000 nucleotides in length, with a preferred range of about 8 to 30nucleotides, and optimally, a range of 10 to 25 nucleotides.

As used herein, a “first region” as it refers to a template nucleicacid, described herein below, means a length of nucleotides sufficientto permit hybridization and extension of a released flap of a secondoligonucleotide wherein the “first region” is complementary to at leasta portion of a released flap of the second oligonucleotide, definedherein. A “first region” is in the range of about 6 nucleotides to about1000 nucleotides in length, with a preferred range of about 8 to 30nucleotides, and optimally, a range of 10 to 25 nucleotides.

As used herein, “extension region” refers to a length of nucleotidessufficient to permit extension of an oligonucleotide (e.g., a firstoligonucleotide or the released flap of a second oligonucleotide) via anucleic acid polymerization activity. An “extension region” of about 1nucleotide to about 1000 nucleotides in length, with a preferred rangeof about 1-100 nucleotides, a more preferred range of 3 to 50, andoptimally, a range of 3-10 nucleotides in length. An “extension region”is of a length that is sufficient such that a cleavage means accordingto the invention will not cleave a downstream oligonucleotide (e.g., asecond oligonucleotide or third oligonucleotide) unless the upstreamprimer (e.g., a first oligonucleotide or the released flap of a secondoligonucleotide, as defined herein) has been extended via polymerizationof a nucleic acid complementary to the extension region such that the 3′end of the primer is close enough to the downstream oligonucleotide(i.e., that hybridizes to the second region) to permit cleavage of theflap (which is the 5′ portion of the downstream oligonucleotide) by thecleavage means.

As used herein, a “second region” as it refers to a target nucleic acid,means a length of nucleotides that is sufficient to permit hybridizationof a second oligonucleotide, wherein the “second region” iscomplementary to at least a portion of a second oligonucleotide, definedherein. A “second region” is in the range of about 6 nucleotides toabout 1000 nucleotides in length, with a preferred range of about 8 to30 nucleotides, and optimally, a range of 10 to 25 nucleotides.

As used herein, a “second region” as it refers to a template nucleicacid, described herein below, means a length of nucleotides that issufficient to permit hybridization of a third oligonucleotide, whereinthe “second region” is complementary to at least a portion of a thirdoligonucleotide, defined herein. A “second region” is in the range ofabout 6 nucleotides to about 1000 nucleotides in length, with apreferred range of about 8 to 30 nucleotides, and optimally, a range of10 to 25 nucleotides.

As used herein, “at least a portion of”, as it refers to a first,second, third or fourth oligonucleotide, means less than 100%, (e.g.,99%, 90%, 75%, 50%, 25% etc . . . ) of the nucleotides of the first,second, third or fourth oligonucleotide.

As used herein, a “template nucleic acid” refers to a polynucleotidewhich comprises in 3′ to 5′ order a first region that is complementaryto at least a portion of the released flap of the secondoligonucleotide, an extension region and a second region that iscomplementary to at least a portion of a third oligonucleotide. A“template nucleic acid” also refers to a polynucleotide which comprisesin 3′ to 5′ order a first region that is complementary to at least aportion of the released flap of the second oligonucleotide, definedherein below, a first extension region, a second region that iscomplementary to at least a portion of a third oligonucleotide, a secondextension region, and a third region that is complementary to at least aportion of a fourth oligonucleotide. The template nucleic acid maycomprise single or double-stranded DNA or RNA or chemical modificationsor unnatural variants of such.

As used herein, “oligonucleotide” refers to a nucleic acid comprising aregion that is complementary to a target nucleic acid sequence and/or atemplate nucleic acid sequence.

As used herein, the term “oligonucleotide” also refers to primers,probes, and oligomer fragments to be detected, and shall be generic topolydeoxyribonucleotides (containing 2-deoxy-D-ribose), topolyribonucleotides (containing D-ribose), and to any other type ofpolynucleotide which is an N-glycoside of a purine or pyrimidine base,or modified purine or pyrimidine bases (including abasic sites). Theterm “oligonucleotide” includes double- and single-strand DNA, as wellas double- and single-strand RNA. The term “oligonucleotide” intends apolynucleotide of genomic DNA or RNA, cDNA, semisynthetic, or syntheticorigin which, by virtue of its synthetic origin or manipulation: (1) isnot associated with all or a portion of the polynucleotide with which itis associated in nature; and/or (2) is linked to a polynucleotide otherthan that to which it is linked in nature. Where the oligonucleotide isused as a primer for the polymerizing activity to polymerize nucleotidesfrom its 3′ end complementary to the extension region, theoligonucleotide also may be referred to as a primer. An“oligonucleotide” according to the invention also refers to peptidenucleic acids (PNA) or hybrids of nucleic acids and peptide nucleicacids.

Oligonucleotides useful in the invention are generally in the range ofabout 8 nucleotides to about 200 nucleotides in length.

A “first oligonucleotide” according to the invention is preferably 6 to100, more preferably 8 to 30 and most preferably 20 nucleotides inlength. A “first” oligonucleotide is at least partially complementary tothe target nucleic acid, and must be complementary to the target nucleicacid at a length of its 3′ terminus sufficient to permit its use as aprimer for nucleic acid synthesis using the target nucleic acid as atemplate.

A “second oligonucleotide” according to the invention is preferably20-120, more preferably 25-45 and most preferably 35 nucleotides inlength. A “second oligonucleotide” comprises a 3′ and a 5′ region. The3′ region of a “second oligonucleotide” is at least partiallycomplementary to the target nucleic acid and is preferably 8-80 and mostpreferably 10-20 nucleotides. A 5′ region of a “second oligonucleotide”is preferably 0 to 80 and most preferably 10 to 20 nucleotides in lengthfor embodiments wherein a duplex structure comprises a preformed flap,or most preferably 0 to 20 nucleotides in length for embodiments whereina flap is formed by the activity of a polymerization means thatdisplaces at least 6 nucleotides. In one embodiment of the invention, a5′ region of a “second oligonucleotide” is at least partiallycomplementary to a region of a target nucleic acid. In anotherembodiment of the invention, the 5′ region of the second oligonucleotideaccording to the invention is not complementary to a target nucleicacid.

A “third oligonucleotide” and “fourth oligonucleotide” according to theinvention are preferably 20-120, more preferably 25-45 and mostpreferably 35 nucleotides in length. A “third oligonucleotide” and“fourth oligonucleotide” comprise a 3′ and a 5′ region. Third and fourtholigonucleotides according to the invention comprise a 3′ region that isat least partially complementary to a region of a template nucleic acidand is preferably 8 to 80 and most preferably 10-20 nucleotides. A 5′region of a “third oligonucleotide” or “fourth oligonucleotide” ispreferably 0 to 80, most preferably 10 to 20 nucleotides in length forembodiments wherein a duplex structure comprises a preformed flap, ormost preferably 0 to 20 nucleotides in length for embodiments wherein aflap is formed by the activity of a polymerization means that displacesat least 6 nucleotides. In an embodiment of the invention, a 5′ regionof a “third oligonucleotide” or “fourth oligonucleotide” is at leastpartially complementary to a region of a template nucleic acid. Inanother embodiment of the invention, the 5′ region of the “thirdoligonucleotide” or “fourth oligonucleotide” according to the inventionis not complementary to a template nucleic acid.

As used herein, an “upstream primer” according to the invention ispreferably 6 to 100, more preferably 8 to 30 and most preferably 20nucleotides in length. An “upstream primer” is at least partiallycomplementary to the target nucleic acid and/or the template nucleicacid at a length of its 3′ terminus sufficient to permit its use as aprimer for nucleic acid synthesis using the target nucleic acid or thetemplate nucleic acid as a template. An “upstream primer” according tothe invention includes a “first oligonucleotide”, as defined herein anda “released flap”, as defined herein.

As used herein, a “downstream oligonucleotide” according to theinvention is preferably 20-120, more preferably 25-45 and mostpreferably 35 nucleotides in length. A “downstream oligonucleotide”comprises a 3′ and a 5′ region. The 3′ region of a downstreamoligonucleotide is at least partially complementary to a target nucleicacid and/or a template nucleic acid. A 5′ region of a “downstreamoligonucleotide” is preferably 0 to 80 nucleotides in length, mostpreferably 10 to 20 nucleotides in length for embodiments wherein aduplex structure comprises a preformed flap, and most preferably 0 to 20nucleotides in length for embodiments wherein a flap is formed by thestrand displacement activity (of at least 6 nucleotides) of apolymerization means. In one embodiment, the 5′ region of a “downstreamoligonucleotide” is at least partially complementary to a region thatmay be upstream of, downstream of, or comprise any of the first orsecond region of a target nucleic acid, or any of the first, second orthird region of a template nucleic acid. In one embodiment the 5′ regionof a “downstream oligonucleotide” is not complementary to a targetnucleic acid or a template nucleic acid. A “downstream oligonucleotide”according to the invention includes a “first” and a “second” downstreamoligonucleotide wherein a “second downstream oligonucleotide” isdownstream of a “first downstream oligonucleotide”. A “downstreamoligonucleotide” according to the invention also includes a second,third or fourth oligonucleotide, as defined herein.

As used herein, “fully complementary” means that 100% of the nucleotidesof an oligonucleotide can hydrogen bond to the correspondingcomplementary nucleotides of the target of template nucleic acid.

As used herein, “at least partially complementary” as it refers to anoligonucleotide, means that less than 100%, (e.g., 99%, 90%, 75%, 50%,25% etc . . . ) of the nucleotides of the oligonucleotide can hybridize(that is form hydrogen bonds) with nucleotides of the target or templatenucleic acid under standard stringent conditions. Where anoligonucleotide is “partially complementary”, the region ofcomplementary nucleotides may or may not be contiguous nucleotides.

For a “first oligonucleotide”, as defined herein, (which also serves asa primer for nucleic acid synthesis), the 5′ region of theoligonucleotide may contain non-complementary nucleotides (with respectto the target nucleic acid), and the 3′ region of the oligonucleotidemust comprise a region of complementarity with the target that issufficient to permit formation of a hybrid with the target nucleic acidand nucleic acid synthesis from the 3′ terminus of the firstoligonucleotide using the target as the complementary strand. Nucleicacid synthesis refers to the addition of at least one nucleotide (forexample a radiolabeled nucleotide (e.g., ³⁵S or ³²P labeleddeoxyribonucleoside triphosphates (dNTPs)), preferably 1-10, and mostpreferably 1-250 nucleotides, to the 3′ end of a nucleic acid molecule(for example as determined by a dideoxy sequencing reaction (accordingto methods well-known in the art) or in a labeling/terminationsequencing reaction using modified T7 DNA polymerase (Sequenase™)(according to methods well known in the art). Alternatively, DNAsynthesis is determined in a 3′ end labeling reaction in the presence ofa radiolabeled dNTP and a polymerase (e.g., the Klenow fragment of E.coli DNA polymerase I) (according to methods well known in the art). Theamount of synthesis can be determined by measuring the size of theradiolabeled fragment by gel electrophoresis. In one embodiment, the 3′region of a first oligonucleotide comprises a region of complementaritywith the target nucleic acid that includes a length of 10 nucleotides orgreater (20, 30, 40, 50, etc.), over contiguous nucleotides (withrespect to the target). In another embodiment, the 3′ region of a firstoligonucleotide comprises a region of complementarity with the targetnucleic acid that includes a sufficient number of non-contiguousnucleotides, that are complementary with the target nucleic acid, topermit formation of a hybrid with the target nucleic acid and nucleicacid synthesis from the 3′ terminus of the first oligonucleotide usingthe target as the complementary strand.

For a “second oligonucleotide”, as defined herein, which does not serveas a primer for nucleic acid synthesis, but rather provides a flap atits 5′ region, “partial complementarity” refers to a region ofnucleotides of non-complementarity with respect to the target nucleicacid, followed by a region of sufficient complementarity to permithydrogen bonding to the target nucleic acid under standard stringentconditions, wherein the second oligonucleotide is capable of forming aduplex and/or cleavage structure according to the invention. In oneembodiment, the region of sufficient complementarity may be 10contiguous nucleotides or longer (e.g., 20, 30, 40, 50, 100, etc.). Inanother embodiment, the region of sufficient complementarity includes asufficient number of non-contiguous nucleotides, that are complementarywith the target nucleic acid, to permit formation of a hybrid with thetarget nucleic acid. The 3′ terminus of the “second oligonucleotide” canbe but is not required to be complementary to the target nucleic acid.Where the 3′ terminus is not complementary to the target nucleic acid(for example, in such instances where the 3′ terminus is labeled and/orserves a function in detection of the hybridized or non/hybridizedoligonucleotide), the region of non-complementary may or may not becontiguous. In one embodiment, the region of non-complementarity may becontiguous for 1 nucleotide, 2 nucleotides, 3, 4, 5 nucleotides, etc.,or over a longer stretch of 10 or greater contiguous nucleotides (20,30, 40, 50, etc.). In another embodiment, the region ofnon-complementarity includes a sufficient number of non-contiguousnucleotides that are non-complementary with the target nucleic acid.

A “first flap”, “second flap”, “third flap” or “fourth flap”, or a“first released flap”, “second released flap”, “third released flap” or“fourth released flap”, according to the invention, is preferably 6 to80 and most preferably 10-25 nucleotides in length.

For a “released flap”, as defined herein, which may serve as a primerfor nucleic acid synthesis when hybridized to the template nucleic acid,the 5′ region of the flap may contain non-complementary nucleotides(with respect to the template), however, the 3′ terminus of the flap mayor may not be complementary with the template. In one embodiment, the 3′terminus of the flap is complementary with the template for a length of10 nucleotides or greater (20, 30, 40, 50, etc.), over contiguousnucleotides. In another embodiment, the 3′ terminus of the flapcomprises a region of complementarity with the template nucleic acidthat includes a sufficient number of non-contiguous nucleotides that arecomplementary to the template nucleic acid. The region ofcomplementarity must include a sufficient number of contiguousnucleotides to permit formation of a hybrid with the template nucleicacid and nucleic acid synthesis from the 3′ terminus of the flap usingthe template as the complementary strand.

For a “third oligonucleotide”, as defined herein, which does not serveas a primer for nucleic acid synthesis, but rather provides a flap atits 5′ region, “partial complementarity” refers to a region of at least10 contiguous nucleotides (20, 30, 40, 50 nucleotides, etc.) ofnon-complementarity with respect to the template nucleic acid, followedby a region of sufficient complementarity to permit hydrogen bonding tothe template nucleic acid under standard stringent conditions. Thisregion of sufficient complementarity may be 10 contiguous nucleotides orlonger (e.g., 20, 30, 40, 50, 100, etc.). The 3′ terminus of the “thirdoligonucleotide” can be but is not required to be complementary to thetemplate nucleic acid. Where the 3′ terminus is not complementary to thetemplate nucleic acid (for example, in such instances where the 3′terminus is labeled and/or serves a function in detection of thehybridized or non/hybridized oligonucleotide), it may benon-complementary for 1 nucleotide, 2 nucleotides, 3, 4, 5 nucleotides,etc., or over a longer stretch of 10 or greater contiguous nucleotides(20, 30, 40, 50, etc.) so long as the complementarity of the thirdoligonucleotide with the template nucleic acid is not disrupted.

A “third oligonucleotide”, as defined herein, may provide a 5′ flapwhich may be cleaved and released from a cleavage structure, and alsomay serve as a primer for nucleic acid synthesis. Where the thirdoligonucleotides serves as a primer for nucleic acid synthesis, the 5′region of the oligonucleotide may contain non-complementary nucleotides(with respect to the template nucleic acid), but the 3′ terminus of theoligonucleotide must be complementary, for a length of 10 nucleotides orgreater (20, 30, 40, 50, etc.), over contiguous nucleotides (withrespect to the template). The region of complementarity with thetemplate must include a sufficient number of contiguous nucleotides topermit formation of a hybrid with the template nucleic acid and nucleicacid synthesis from the 3′ terminus of the third oligonucleotide usingthe template as the complementary strand.

For a “fourth oligonucleotide”, as defined herein, which does not serveas a primer for nucleic acid synthesis, but rather provides a flap atits 5′ region, “partial complementarity” refers to a region of at least10 contiguous nucleotides (20, 30, 40, 50 nucleotides, etc.) ofnon-complementarity with respect to the template nucleic acid, followedby a region of sufficient complementarity to permit hydrogen bonding tothe template nucleic acid under standard stringent conditions. Thisregion of sufficient complementarity may be 10 contiguous nucleotides orlonger (e.g., 20, 30, 40, 50, 100, etc.). The 3′ terminus of the “fourtholigonucleotide” can be but is not required to be complementary to thetemplate nucleic acid. Where the 3′ terminus is not complementary to thetemplate nucleic acid (for example, in such instances where the 3′terminus is labeled and/or serves a function in detection of thehybridized or non/hybridized oligonucleotide), it may benon-complementary for 1 nucleotide, 2 nucleotides, 3, 4, 5 nucleotides,etc., or over a longer stretch of 10 or greater contiguous nucleotides(20, 30, 40, 50, etc.) so long as the complementarity of the fourtholigonucleotide with the template nucleic acid is not disrupted.

As used herein, “mixing” means combining, in any order.

As used herein, “conditions which permit formation of a duplex” refer toa buffer (i.e., of a specified salt and organic solvent concentration),a temperature, an incubation time, and the concentrations of thecomponents of the duplex (for example a target nucleic acid, a firstoligonucleotide and a second oligonucleotide) that are possible andpreferably optimal for the formation of a duplex of the invention. Forexample, in one embodiment of the invention, under “conditions whichpermit formation of a duplex”, a target nucleic acid, a firstoligonucleotide and a second oligonucleotide will hybridize such thatthe 5′ region of the second oligonucleotide is a flap and the extensionregion of the target nucleic acid is not hybridized to a first, second,third or fourth oligonucleotide (FIG. 2).

As used herein, “duplex” refers to a complex comprising a target ortemplate nucleic acid, a first oligonucleotide or an upstream primer andat least a 3′ region of a second oligonucleotide or a downstreamoligonucleotide, wherein the complementary nucleotide bases of thetarget or template nucleic acid and each of the first oligonucleotide orupstream primer and, at least a 3′ region of a second oligonucleotide ora downstream oligonucleotide are hybridized due to the formation ofhydrogen bonds and where the “at least a portion of the extensionregion” is not hydrogen bonded to a first, second, third, fourth ordownstream oligonucleotide, as defined herein.

As used herein, “first duplex” refers to a complex comprising a targetor template nucleic acid, a first oligonucleotide and at least a 3′region of a second oligonucleotide, wherein the complementary nucleotidebases of the target or template nucleic acid and each of the firstoligonucleotide and, at least a 3′ region of a second oligonucleotideare hybridized due to the formation of hydrogen bonds, and wherein the5′ region of a second oligonucleotide and the extension region of thetarget nucleic acid are not complementary and thus do not form a hybridin the duplex. The extension region may thus be single-stranded.

As used herein, “first duplex” also refers to a complex comprising atemplate nucleic acid, an upstream primer and at least a 3′ region of adownstream oligonucleotide, wherein the complementary nucleotide basesof the template nucleic acid and each of the upstream primer and atleast a 3′ region of the downstream oligonucleotide are hybridized dueto the formation of hydrogen bonds, wherein the 5′ region of thedownstream oligonucleotide is a flap.

As used herein, “first duplex” also refers to a complex comprising atemplate nucleic acid, an upstream primer, at least a 3′ region of afirst downstream oligonucleotide and at least a 3′ region of a seconddownstream oligonucleotide, wherein the complementary nucleotide basesof the template nucleic acid and each of the upstream primer, the atleast a 3′ region of the first downstream oligonucleotide, and the atleast a 3′ region of the second downstream oligonucleotide arehybridized due to the formation of hydrogen bonds, wherein the 5′regions of each of the first and second downstream oligonucleotides are,independently, flaps.

As used herein, “second duplex” refers to a complex comprising atemplate nucleic acid, the released flap of the second oligonucleotide,and at least a 3′ region of a third oligonucleotide, wherein thecomplementary nucleotide bases of the template nucleic acid and each ofthe released flap of the second oligonucleotide, and the at least a 3′region of a third oligonucleotide are hybridized due to the formation ofhydrogen bonds with the template nucleic acid.

As used herein, “second duplex” refers to a complex comprising atemplate nucleic acid, the released flap of the second oligonucleotide,and at least a 3′ region of a third oligonucleotide, wherein thecomplementary nucleotide bases of the target nucleic acid and each ofthe released flap of the second oligonucleotide, and the at least a 3′region of a third oligonucleotide are hybridized to the template due tothe formation of hydrogen bonds, and wherein the 5′ region of the thirdoligonucleotide and the extension region of the template nucleic acidare not complementary and thus do not form a hybrid in the duplex. Theextension region thus may be single-stranded.

As used herein, “second duplex” also refers to a complex comprising atemplate nucleic acid, an upstream primer and at least a 3′ region of adownstream oligonucleotide, wherein the complementary nucleotide basesof the template nucleic acid and each of the upstream primer and atleast a 3′ region of the downstream oligonucleotide are hybridized dueto the formation of hydrogen bonds, wherein the 5′ region of thedownstream oligonucleotide is a flap.

As used herein, “second duplex” also refers to a complex comprising atemplate nucleic acid, the released flap of the second oligonucleotide,at least a 3′ region of a third oligonucleotide, and at least a 3′region of a fourth oligonucleotide wherein the complementary nucleotidebases of the template nucleic acid and each of the released flap of thesecond oligonucleotide, the at least a 3′ region of a thirdoligonucleotide, and the at least a 3′ region of a fourtholigonucleotide are hybridized to the template due to the formation ofhydrogen bonds.

As used herein, “second duplex” also refers to a complex comprising atemplate nucleic acid, the released flap of the second oligonucleotide,at least a 3′ region of a third oligonucleotide, and at least a 3′region of a fourth oligonucleotide wherein the complementary nucleotidebases of the template nucleic acid and each of the released flap of thesecond oligonucleotide, the at least a 3′ region of a thirdoligonucleotide, and the at least a 3′ region of a fourtholigonucleotide are hybridized to the template due to the formation ofhydrogen bonds, and wherein the 5′ region of the third oligonucleotideand the 5′ region of the fourth oligonucleotide are not hybridized to,respectively, the first and second extension regions of the templatenucleic acid. Thus, the 3′ region of the third oligonucleotide, the 3′region of the fourth oligonucleotide, and the first and second extensionregions may be single-stranded in the duplex.

As used herein, “second duplex” also refers to a complex comprising atemplate nucleic acid, an upstream primer, at least a 3′ region of afirst downstream oligonucleotide and at least a 3′ region of a seconddownstream oligonucleotide, wherein the complementary nucleotide basesof the template nucleic acid and each of the upstream primer, the atleast a 3′ region of the first downstream oligonucleotide, and the atleast a 3′ region of the second downstream oligonucleotide arehybridized due to the formation of hydrogen bonds, wherein the 5′regions of each of the first and second downstream oligonucleotides are,independently, flaps.

A “flap” or a “arm” of a branched DNA or DNA/RNA hybrid, refers to a 5′polynucleotide that is not hydrogen-bonded to the branched DNA or hybridDNA/RNA, but is phosphate-bonded to a hydrogen-bonded member of thebranched DNA or DNA/RNA hybrid. A flap thus is a nucleic acid strandwhich hangs off of (i.e., is the branch off of) a double strandedportion of the structure. A “flap” of a cleavage structure according tothe invention is preferably about 1-80 nucleotides, more preferablyabout 5-25 nucleotides and most preferably about 10-20 nucleotides, andis preferably cleaved at a position located at the phosphate positionedat the “elbow” of the branched structure or at any of one to tenphosphates located proximal and/or distal to the elbow of the flapstrand. The cleavage position depends on whether the polymerizationmeans possesses or lacks endonucleolytic activity. As used herein,“elbow” refers to the phosphate bond between the first single strandednucleotide of the 5′ flap and the first double stranded (e.g.,hybridized to the target or template nucleic acid) nucleotide. A “flap”,according to the invention can be labeled with a detectable label. A“flap” or “arm” according to the invention is cleaved by a cleavagemeans when it is part of a “cleavage structure”, as defined herein, andis released to form a “released arm” which may be single-stranded (i.e.,have no double-stranded structure), or may simple be a single strandnucleic acid which contains internal complementarity or is duplexed withanother nucleic acid strand that is not phosphate bonded to the branchedDNA or DNA/RNA hybrid. In those instances wherein the “released flap”serves as a primer for nucleic acid synthesis, the “released flap” ispreferably 6 to 80 and most preferably 10-25 nucleotides in length. Inthose embodiments wherein the flap is strand displaced (i.e., is not apreformed flap), there may be less than a substantial overlap betweenthe upstream primer and the downstream oligonucleotides. As used herein,“less than a substantial overlap” means an overlap length that permitsformation of a “released flap” that is at least 6 nucleotides in lengthand can serve as a primer for nucleic acid synthesis.

The term “single strand”, with respect to a nucleic acid, refers to onepolynucleotide strand which may not be hydrogen-bonded to any othernucleic acid, or it may be hydrogen-bonded internally to itself (to forma secondary or tertiary structure) or to another nucleic acid molecule.

The term “single-stranded”, with respect to a nucleic acid, refers to apolynucleotide strand which is not hydrogen-bonded to another nucleicacid, and which preferably contains no or little (less than 10%, forexample 9%, 5%, 4%, etc. . . .) internal complementarity.

As used herein, “extension” refers to the addition of nucleosidetriphosphates to the 3′ end of a first oligonucleotide or the releasedsingle-stranded arm of the second oligonucleotide in a conventional DNApolymerization reaction. Thus, the 3′ end of the first oligonucleotideand the 3′ end of the released single-stranded arm of the secondoligonucleotide are not blocked, and are also referred to herein asprimers.

Generally the 3′ terminus of the template, and the second, third andfourth oligonucleotides will be “blocked” to prohibit creation of anextension product. “Blocking” can be achieved by using non-complementarybases or by adding a chemical moiety such as biotin or a phosphate groupto the 3′ hydroxyl of the last nucleotide. Blocking can also be achievedby removing the 3′-OH or by using a nucleotide that lacks a 3′-OH suchas dideoxynucleotide, or by other methods known to one skilled in theart.

As used herein, “nucleic acid polymerization activity” refers to anenzyme that catalyzes the polymerization of nucleoside triphosphates.Generally, the enzyme will initiate synthesis at the 3′-end of theprimer annealed to the target sequence, and will proceed in the5′-direction along the template, and if possessing a 5′ to 3′ nucleaseactivity, hydrolyze intervening, annealed probe to release both labeledand unlabeled probe fragments, until synthesis terminates. Known DNApolymerases include, for example, E. coli DNA polymerase I, T7 DNApolymerase, Thermus thermophilus (Tth) DNA polymerase, Bacillusstearothermophilus DNA polymerase, Thermococcus litoralis DNApolymerase, Thermus aquaticus (Taq) DNA polymerase and Pyrococcusfuriosus (Pfu) DNA polymerase. If the nucleic acid template is RNA, then“nucleic acid polymerization activity” refers to an RNA-dependentpolymerization activity, such as reverse transciptase.

As used herein, “5′ to 3′ exonuclease activity” or “5′→3′ exonucleaseactivity” refers to that activity of a template-specific nucleic acidpolymerase e.g. a 5′→3′ exonuclease activity traditionally associatedwith some DNA polymerases whereby mononucleotides or oligonucleotidesare removed from the 5′ end of a polynucleotide in a sequential manner,(i.e., E. coli DNA polymerase I has this activity whereas the Klenow(Klenow et al., 1970, Proc. Natl. Acad. Sci., USA, 65:168) fragment doesnot, (Klenow et al., 1971, Eur. J. Biochem., 22:371)), orpolynucleotides are removed from the 5′ end by an endonucleolyticactivity that may be inherently present in a 5′ to 3′ exonucleaseactivity.

As used herein, the phrase “substantially lacks 5′ to 3′exonucleaseactivity” or “substantially lacks 5′→3′ exonuclease activity” meanshaving less than 10%, 5%, 1%, 0.5%, or 0.1% of the activity of a wildtype enzyme. The phrase “lacking 5′ to 3′ exonuclease activity” or“lacking 5′→3′ exonuclease activity” means having undetectable 5′ to 3′exonuclease activity or having less than about 1%, 0.5%, or 0.1% of the5′ to 3′ exonuclease activity of a wild type enzyme. 5′ to 3′exonuclease activity may be measured by an exonuclease assay whichincludes the steps of cleaving a nicked substrate in the presence of anappropriate buffer, for example 10 mM Tris-HCl (pH 8.0), 10 mM MgCl₂ and50 μg/ml bovine serum albumin) for 30 minutes at 60° C., terminating thecleavage reaction by the addition of 95% formamide containing 10 mM EDTAand 1 mg/ml bromophenol blue, and detecting nicked or un-nicked product.

According to methods of the invention that include two polymerizationsteps, each of the polymerization steps can be performed by the samenucleic acid polymerization activity or by different nucleic acidpolymerization activities.

As used herein, “polymerization”, “polymerization activity” or“polymerase acitivity” refers to the addition of nucleosidetriphosphates to the 3′ end of an oligonucleotide wherein the 3′ end ofthe oligonucleotide is not blocked.

As used herein, “nucleoside” refers to any purine or pyrimidine base, ormodified purine or pyrimidine base, linked to a sugar (e.g.2-deoxyribose in DNA or ribose in RNA).

As used herein, “nucleotide” refers to any purine or pyrimidine base, ormodified purine or pyrimidine base, linked to a sugar, wherein the sugaris linked to a phosphate group.

As used herein, “polymerase chain reaction” or “PCR” refers to an invitro method for amplifying a specific polynucleotide template sequence.The PCR reaction involves a repetitive series of temperature cycles andis typically performed in a volume of 50-100 μl. The reaction mixcomprises dNTPs (each of the four deoxyribonucleotides dATP, dCTP, dGTP,and dTTP), primers, buffers, DNA polymerase, and polynucleotidetemplate. One PCR reaction may consist of 5 to 100 “cycles” ofdenaturation and synthesis of a polynucleotide molecule. The PCR processis described in U.S. Pat. Nos. 4,683,195 and 4,683,202, the disclosuresof which are incorporated herein by reference.

As used herein, “polymerase chain reaction conditions” or “PCRconditions” refer to a buffer, a set of temperature incubation steps andtimes that are possible and preferably optimal for conducting PCR. Theset of temperature incubation steps and times preferably allow fordenaturation, annealing, and extension of the nucleic acids of theinvention. PCR reaction conditions are known in the art and describedherein.

A “nucleic acid amplification reaction” means an increase in number of aparticular nucleic acid sequence and may be accomplished, withoutlimitation, by the in vitro methods of polymerase chain reaction orligase chain reaction.

As used herein, “nucleic acid amplification reaction conditions” referto a buffer, a set of temperature incubation steps and times that arepossible and preferably optimal for conducting amplification of anucleic acid. Amplification means an increase in the number of aparticular nucleic acid sequence and may be accomplished, withoutlimitation, by the in vitro methods of polymerase chain reaction orligase chain reaction. Such reaction conditions are known in the art andare described herein. Nucleic acid reaction conditions encompass PCRconditions.

A “nucleic acid amplification reaction mixture” refers to a mixturecomprising the components necessary for nucleic acid amplification, suchas buffer, dNTPs, water, a target nucleic acid, and a polymerase.Depending on methods for nucleic acid amplification, the necessarycomponents may differ. For example, ligase chain reactions require thepresence of a ligase, which may not be needed in certain polymerasechain reactions.

As used herein, a “cleavage structure” refers to a polynucleotidestructure comprising at least a duplex nucleic acid having a flap. A“cleavage structure” according to the invention preferably comprises atarget nucleic acid sequence or a template nucleic acid sequence, andalso includes an upstream oligonucleotide that is at least partially andmay be fully complementary to the target sequence (for example, A inFIG. 2 or F in FIG. 5), and a downstream oligonucleotide that iscomplementary to the target sequence and comprises a flap (for exampleFC or FH in FIG. 2 or FH1 or FH2 in FIG. 5). In one embodiment, a “firstcleavage structure” is formed by polymerization from the 3′ end of theupstream oligonucleotide (primer) through the extension region to theduplex formed by hydrogen bonding of the downstream oligonucleotide andthe target or template nucleic acid (i.e., at the junction of the duplexand the flap). In another embodiment, a “second cleavage structure” isformed by polymerization from the 3′ end of the upstream oligonucleotide(primer) (i.e., the released arm of the second oligonucleotide) throughthe extension region to the duplex formed by hydrogen bonding of thedownstream third oligonucleotide (or first downstream oligonucleotide)and the template nucleic acid (i.e., at the junction of the duplex andthe flap). A “second cleavage structure” also refers to a cleavagestructure wherein polymerization has occurred from the 3′ end of thesecond oligonucleotide to the duplex formed by hydrogen bonding of thedownstream fourth oligonucleotide (or second downstream oligonucleotide)and the template nucleic acid (i.e., at the junction of the duplex andthe flap). Preferably, the 3′ terminus of the upstream oligonucleotideis blocked; blocking the terminus prevents extension of the 3′ end ofthe upstream oligonucleotide.

In a “cleavage structure” according to the invention, polymerization(i.e., from the 3′ end of a primer through an extension region to theduplex formed by hydrogen bonding of the downstream oligonucleotide tothe target or template nucleic acid) may continue either partially orcompletely through the extension region.

Alternatively, in some reactions the polymerization may continue eitherpartially or completely through regions 5′ to the extension region(e.g., second region of a template nucleic acid or target nucleic acid).In such reactions the downstream probe (e.g., second or thirdoligonucleotide) may be cleaved such that the downstream probe no longerstable hybridizes with the template or target nucleic acid.

In those reactions where polymerization continues partially through theextension region, the newly polymerized nucleic acid must reach “closeenough” to the duplex formed by the hydrogen bonding of the downstreamoligonucleotide and target or template nucleic acid (i.e., the three-wayflap junction region) so as to form a “cleavage structure”) so as topermit cleavage and release of the flap from the cleavage structure.That is, the length of extension region which is rendereddouble-stranded via polymerization from the 3′ end of the upstreamprimer must be “sufficient” to form a “cleavage structure” and to permitcleavage of the flap by the cleavage means.

Thus, the term “close enough” for the newly polymerized nucleic acid'sproximity to the three-way junction formed by the flap, the downstreamoligonucleotide to which the flap is phosphate bonded, and the target ortemplate nucleic acid to which the oligonucleotide is hydrogen bonded,as well as the term “length of extension region” “sufficient to permitcleavage”, refers to a nucleotide distance (between the 3′ end of thenewly-synthesized strand and the first nucleotide that is duplexed inthe three-way junction) of no greater than 50 nucleotides and preferably10 nucleotides or less, such as 6 nucleotides, 4 nucleotides, 2nucleotides, 1 nucleotide, or no nucleotides. However, should a flapcleavage enzyme be discovered or developed which cleaves in the presenceof a gap of greater than 50 nucleotides, (e.g., 100 nucleotides or more)the invention contemplates encompassing such a gap length.

As used herein, the alternative term “adjacent to” also may be used.

In those reactions where polymerization of a newly synthesized nucleicacid continues completely through the extension region, the distancereferred to above would of course be no nucleotides. Alternatively, insome reactions where the cleavage structure contains a template nucleicacid comprising first and second extension regions, a primer (cleavedand released flap), and third and fourth oligonucleotides, where thethird oligonucleotide forms a hybrid with the region of the templatestrand that is between the first and second extension regions and thefourth oligonucleotide forms a hybrid with the template regiondownstream of the second extension region, the polymerase activity maycontinue through the first extension region and displace the thirdoligonucleotide as it continues through the template second region,fully displacing the third oligonucleotide, and continuing synthesispartially or completely through the second extension region until the 3′end of the newly synthesized nucleic acid comes “close enough” to thethree-way junction of the 5′ flap of the fourth oligonucleotide, the 3′portion of the fourth oligonucleotide which is hydrogen bonded to thetemplate nucleic acid to permit cleavage and release of the flap fromthe fourth oligonucleotide.

Thus, a “cleavage structure” according to the invention may be anupstream primer which is hybridized to a template nucleic acid, and adownstream oligonucleotide which includes a flap which is the 5′ portionof the downstream oligonucleotide. In this embodiment of the invention,the upstream and downstream oligonucleotides (e.g., A and FC of FIG. 2)may be non-overlapping. In those embodiments wherein the flap is stranddisplaced (i.e., is not a preformed flap), there may be less than asubstantial overlap between the upstream and downstreamoligonucleotides. The invention also provides for upstream anddownstream oligonucleotides wherein one or both of the oligonucleotidesis completely complementary to an extension region.

In forming a “cleavage structure” according to the invention,transitional structures may be formed, such as a polynucleotidestructure comprising a loop, a pseudo-Y structure, a single-strandedbubble, a D-loop, a nick or a gap.

A “cleavage structure”, as used herein, includes a 5′ flap and would notencompass a structure which does not include a 5′ flap, for example, adouble stranded nucleic acid which contains only a 3′ flap. As usedherein, a “cleavage structure” comprises ribonucleotides ordeoxyribonucleotides and thus can be RNA or DNA.

As used herein a “cleavage means” refers to an agent, preferably anenzyme, that is specific for, that is, cleaves a cleavage structureaccording to the invention.

In one embodiment of the invention wherein the cleavage means is used incombination with a nucleic acid polymerization activity that has stranddisplacement activity, or is itself a nucleic acid polymerizationactivity that has strand displacement activity, the cleavage means willnot cleave a cleavage structure unless the cleavage product, that is,the released flap, is of a length that is sufficient to permithybridization of the released flap to a template nucleic acid andextension of the released flap by polymerization.

The term “cleavage means” includes an enzyme that possesses 5′endonucleolytic activity for example a DNA polymerase, e.g. DNApolymerase I from E. coli, and DNA polymerase from Thermus aquaticus(Taq), Thermus thermophilus (Tth), and Thermus flavus (Tfl). The term“cleavage means” includes agents that cleave a cleavage structureaccording to the invention comprising an extension region singlestranded gap (that is a portion of the extension region that isunhybrized to an upstream primer and/or a downstream probe) of 0-2nucleotides, 2-20 nucleotides, 20-50 nucleotides or more than 50nucleotides. The term “cleavage means” also embodies FEN nucleases. Theterm “FEN nuclease” encompasses an enzyme that possesses 5′ exonucleaseand/or an endonuclease activity. The term “FEN nuclease” also embodies a5′ flap-specific nuclease. The term “cleavage means” includes a FENnuclease that cleaves a cleavage structure according to the inventioncomprising an extension region single stranded gap (that is a portion ofthe extension region that is unhybrized to an upstream primer and/or adownstream probe) of 0-2 nucleotides, 2-20 nucleotides, 20-50nucleotides or more than 50 nucleotides.

A “cleavage means” according to the invention includes but is notlimited to a FEN nuclease enzyme derived from Archaeglobus fulgidus,Methanococcus jannaschii, Pyrococcus furiosus, human, mouse or Xenopuslaevis. A nuclease according to the invention also includesSaccharomyces cerevisiae RAD27, and Schizosaccharomyces pombe RAD2, PolI DNA polymerase associated 5′ to 3′ exonuclease domain, (e.g. E. coli,Thermus aquaticus (Taq), Thermus flavus (Tfl), Bacillus caldotenax(Bca), Streptococcus pneumoniae) and phage functional homologs of FENincluding but not limited to T5 5′ to 3′ exonuclease, T7 gene 6exonuclease and T3 gene 6 exonuclease. Preferably, only the 5′ to 3′exonuclease domains of Taq, Tfl and Bca FEN nuclease are used. A“cleavage means” according to the invention also includes an agent,preferably an enzyme, that cleaves a cleavage structure according to theinvention comprising an RNA/DNA complex wherein the RNA is the templateor target nucleic acid. The term “cleavage means” does not include RNAseH.

“Cleavage means” also includes enzymes that can cleave a cleavagestructure comprising an upstream oligonucleotide (for example A, FIG. 2)and one or more downstream oligonucleotides (for example (FC, FIG. 2)wherein polymerization has occurred from the 3′ end of the upstreamoligonucleotide such that the extended 3′ end of the upstreamoligonucleotide is adjacent to the flap of the downstreamoligonucleotide (for example F, FIG. 2).

As used herein, “adjacent” refers to separated by less than 20nucleotides, e.g., 15 nucleotides, 10 nucleotides, 5 nucleotides, or 0nucleotides.

“Cleavage means” also includes enzymes that can cleave a cleavagestructure comprising an upstream oligonucleotide (for example A, FIG. 2)and one or more downstream oligonucleotides (for example (FC, FIG. 2)wherein polymerization has occurred from the 3′ end of the upstreamoligonucleotide such that the extended 3′ end of the upstreamoligonucleotide is less than 50 nucleotides from the flap of thedownstream oligonucleotide (for example F, FIG. 2). According to thisembodiment of the invention, the distance between the 3′ end of theupstream oligonucleotide across the extension region and sufficientlyclose to the junction of the flap of the downstream oligonucleotide isof a length that permits a sufficient amount of polymerization to occurfrom the 3′ end of the upstream oligonucleotide to form a cleavagestructure according to the invention.

According to methods of the invention that include two cleavage steps,each of the cleavage steps can be performed by the same cleavage meansor by a different cleavage means.

A “cleavage means” according to the invention can be a single enzymethat possesses both polymerase and nuclease activity or an enzyme thatpossesses nuclease activity but lacks polymerase activity.

As used herein, “permitting” means allowing a reaction to proceed suchthat a duplex or a second duplex, as defined herein, is formed if all ofthe components required for duplex formation (e.g., the first and secondoligonucleotide and the target nucleic acid, or the released flap of thesecond oligonucleotide, the template nucleic acid and the thirdoligonucleotide) are present. “Permitting” also means adding anyrequired components (e.g., the released flap of the secondoligonucleotide) to a mixture comprising the template and the thirdoligonucleotide, and allowing the reaction to proceed such that a“second duplex”, as defined herein, is formed. “Permitting” also meansadding any required components (e.g., the released flap of the secondoligonucleotide) to a mixture comprising the template the thirdoligonucleotide, and the fourth oligonucleotide, and allowing thereaction to proceed such that a “second duplex”, as defined herein, isformed. “Permitting” also means adding any required components (e.g.,template and third oligonucleotide) to the released flap of the secondoligonucleotide and allowing the reaction to proceed such that a duplexor a second duplex, as defined herein, is formed.

As used herein, “detecting a target nucleic acid sequence” or “measuringa target nucleic acid sequence” refers to determining the presence of aparticular target nucleic acid sequence in a sample or determining theamount of a particular target nucleic acid sequence in a sample as anindication of the presence of a target nucleic acid sequence in asample. The amount of a target nucleic acid sequence that can bemeasured or detected is preferably about 1 molecule to 10²⁰ molecules,more preferably about 100 molecules to 10¹⁷ molecules and mostpreferably about 1000 molecules to 10¹⁴ molecules. According to oneembodiment of the invention, the detected nucleic acid is derived fromthe labeled 5′ end of at least one of a first, second or third flaps,defined herein (for example F in FIGS. 2 and 5). According to thepresent invention, a label is attached to the 5′ end of at least one ofa second, third or fourth oligonucleotide, defined herein (for exampleFC, FH1 or FH2 in FIGS. 2 and 5). Alternatively, a label is attached tothe 3′ end at least one of a second, third or fourth oligonucleotide,defined herein (for example FC, FH1 or FH2 in FIGS. 2 and 5) and aquencher is attached to the 5′ end at least one of a second, third orfourth oligonucleotide. According to the invention, a label may beattached to the 3′ end at least one of a second, third or fourtholigonucleotide comprising a cleavage structure according to theinvention. In another embodiment, the detected nucleic acid is derivedfrom the unlabeled released flap and the released flap is detected bygel electrophoresis or by hybridization according to methods well knowin the art.

In another embodiment, the detected nucleic acid is derived from afragment of the third oligonucleotide, defined herein (for example H inFIG. 17). According to this embodiment, a label is operably coupled tothe 3′ end, 5′ end or an internal region of a third oligonucleotide anda quencher is operably coupled to a template nucleic acid.

In yet another embodiment, the detected nucleic acid is derived from afragment of a template nucleic acid. According to the present invention,a label is operably coupled to the 5′ end, 3′ end or an internal regionof the template nucleic acid and a quencher is operably coupled to athird oligonucleotide. A quencher is operably coupled to anoligonucleotide of the invention so as to inhibit the detectable signalwhen the template nucleic acid and third oligonucleotide are hybridizedto each other. In one embodiment, the label is one member or moiety of apair of interactive labels. In this embodiment, the pair of interactivelabels interact when the third oligonucleotide and template nucleic acidhybridize and produce a detectable signal, e.g., increase influorescence, when the third oligonucleotide and template nucleic acidare not hybridized, e.g., after the third oligonucleotide is cleaved anddisassociates from the template nucleic acid.

According to the invention, an oligonucleotide according to theinvention (e.g., a first, second, third fourth oligonucleotide or atemplate nucleic acid) can be labelled by attaching a label to the 5′end, the 3′ end or by labelling the oligonucleotide internally.

In another embodiment, the probe is labeled with a pair of interactivelabels (e.g., a FRET or non-FRET pair) positioned to permit theseparation of the labels during oligonucleotide probe unfolding (e.g.,for example due to hybridization to a target or template nucleic acid,or due to a change in the secondary structure of the probe, as definedhereinbelow, or hydrolysis.

In another embodiment, the probe and template nucleic acid are eachlabeled with one member of a pair of interactive labels (e.g., a FRET ornon-FRET pair). The members of the interactive pair of labels interactwhen the probe (e.g., third oligonucleotide) and the template nucleicacid hybridize. Separation of the probe and template nucleic acid (e.g.,cleavage of the probe) generates a detectable signal (e.g.fluorescence). The pair of interactive labels may be placed anywherealong the third oligonucleotide and template nucleic acid so long as thepair of interactive labels interact when hybridized and produce adetectable signal when they are not hybridized

As used herein “detection of a signal generated by the release of athird oligonucleotide” refers to determining the presence of a cleavedlabeled third oligonucleotide in a sample or determining the amount ofthe cleaved labeled third oligonucleotide. Methods well known in the artand described herein can be used to detect or measure the release of thelabelled third oligonucleotide. The presence or amount of a cleavedlabelled third oligonucleotide may be determined by detecting a changein fluorescence. For example, upon cleavage of the third oligonucleotideand separation of the pair of interactive labels present on the thirdoligonucleotide and a template nucleic acid. The detection methodsdescribed herein are operative for detecting a released thirdoligonucleotide wherein any amount of a released third oligonucleotideis detected whether that be a small or large proportion of the releasedthird oligonucleotides generated in the reaction. A method of detectingor measuring release of the third oligonucleotide will be appropriatefor measuring or detecting the labelled moiety that is present on thereleased third oligonucleotide or on the template nucleic acid.

As used in at least one embodiment, “detecting at least one of thereleased first and second flaps” or “detecting release of at least oneof the first, second and third flaps” refers to determining the presenceof a labelled or unlabeled flap in a sample or determining the amount ofa labelled or unlabeled flap in a sample. Methods well known in the artand described herein can be used to detect or measure release oflabelled or unlabeled flaps. The detection methods described herein areoperative for detecting a flap wherein any amount of a flap is detectedwhether that be a small or large proportion of the flaps generated inthe reaction. A method of detecting or measuring release of labelledflaps will be appropriate for measuring or detecting the labelled moietythat is present on the labelled flap. Methods of detecting or measuringrelease of unlabeled flaps include, for example, gel electrophoresis orby hybridization, according to methods well known in the art. Thedetection methods described herein are operative when as little as 1 or2 molecules (and up to 1 or 2 million, for example 10, 100, 1000,10,000, 1 million) of released flap are detected.

As used herein, “labelled flaps” refers to cleaved mononucleotides orsmall oligonucleotides or oligonucleotides derived from the labelledcleavage structure according to the invention wherein the cleavedoligonucleotides are preferably between about 6-80 nucleotides, and morepreferably between 10-25 nucleotides, which are cleaved from a cleavagestructure by a nuclease and can be detected by methods well known in theart and described herein.

In a preferred embodiment, the first and second flaps comprise anidentical sequence.

In another preferred embodiment, the first and second flaps comprisenon-identical sequences.

In another preferred embodiment, the first oligonucleotide and thesecond oligonucleotide hybridize to non-overlapping regions of thetarget nucleic acid, and the released flap of the second oligonucleotideand the third oligonucleotide hybridize to non-overlapping regions ofthe template nucleic acid.

In a preferred embodiment, the first, second and third flaps comprise anidentical sequence.

In another preferred embodiment, the first and second flaps comprise anidentical sequence.

In another preferred embodiment, the first and third flaps comprise anidentical sequence.

In another preferred embodiment, the first flap comprises a sequencethat is not identical to either of the second or third flaps.

In another preferred embodiment, the first and second oligonucleotideshybridize to non-overlapping regions of the target nucleic acid, andeach of the released first flap of the second oligonucleotide, the thirdoligonucleotide and the fourth oligonucleotide hybridize tonon-overlapping regions of the template nucleic acid.

As used herein, “non-overlapping” means that if two oligonucleotides arehybridized to the target (or template) nucleic acid, then the twooligonucleotides will not compete with each other with respect tohybridization with the target (or template) nucleic acid. Thus, the tworespective hybridization regions of the target (or template) nucleicacid do not involve one or more nucleotides in common.

In another preferred embodiment, the cleavage means comprises a 5′nuclease activity for which cleavage of a flap from a cleavage structureis dependent upon the formation of duplex DNA the site of cleavage.

In another preferred embodiment, the cleavage means comprises a FEN-1nuclease.

In another preferred embodiment, the polymerization activity comprisesstrand displacement activity.

Nucleic acid polymerases exhibiting strand displacement activity anduseful according to the invention include but are not limited toarchaeal DNA polymerases with “temperature activated” stranddisplacement activity (exo plus and exo minus versions of Vent, DeepVent, Pfu, JDF-3, KOD (LTI's tradename Pfx), Pwo, 9 degrees North,Thermococcus aggregans, Thermococcus gorgonarius), and eubacterial DNApolymerases with strand displacement activity (exo minus Bst, exo minusBca, Genta, Klenow fragment, exo minus Klenow fragment exo minus T7 DNApolymerase (Sequenase).

In another preferred embodiment, the method is carried out isothermally.

As used herein, “isothermally” refers to a temperature that supports,and is preferably optimal, for the activity of a cleavage means and apolymerization means according to the invention.

In another preferred embodiment, the method is carried out under aseries of reaction conditions.

As used herein, “a series of reaction conditions” refers to two or moretemperature incubations that are optimal for the steps of duplexformation, polymerization and cleavage according to the invention. Forexample, a series of reaction conditions include PCR reaction andnucleic acid reaction conditions.

In another preferred embodiment, a single enzyme comprises apolymerization activity and a cleavage means.

In another preferred embodiment, a single enzyme comprising apolymerization activity and a cleavage means is selected from the groupconsisting of E. coli DNA polymerase I, T7 DNA polymerase, Tth DNApolymerase, or Taq DNA polymerase.

In another preferred embodiment, a first enzyme comprises apolymerization activity and a second enzyme comprises a cleavage means.

In another preferred embodiment, the first enzyme comprises Thermusthermophilus DNA pol or Reverse transcriptase and the second enzymecomprises a FEN-1 nuclease.

Further features and advantages of the invention will become more fullyapparent in the following description of the embodiments and drawingsthereof, and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a representation of linear amplification of a template nucleicacid sequence.

FIG. 2 is a representation of a linear amplification of a target nucleicacid sequence comprising a duplex structure comprising a preformed flap.

FIG. 3 is a representation of a linear amplification of a target nucleicacid sequence wherein a flap is formed by the activity of apolymerization means with strand displacement activity.

FIGS. 4 a and 4 b are representations of an exponential amplification ofa template nucleic acid sequence.

FIG. 5 is a representation of an exponential amplification of a targetnucleic acid sequence comprising a duplex structure comprising apreformed flap.

FIG. 6 is a representation of an exponential amplification of a targetnucleic acid sequence wherein a flap is formed by the activity of apolymerization means with strand displacement activity.

FIG. 7 is a diagram illustrating a probe comprises secondary structurethat changes upon binding to a target or template nucleic acid sequence,wherein the probe further comprises a binding moiety and/or a tag.

FIG. 8 demonstrates FEN nuclease cleavage structures.

FIG. 9 demonstrates secondary structures.

FIG. 10 is a representation of a safety pin probe.

FIG. 11 is a representation of a scorpion probe.

FIG. 12 is a representation of a sunrise/amplifluor probe.

FIG. 13 a is a graph demonstrating the difference in light absorbance ofdouble-stranded versus single-stranded DNA.

FIG. 13 b is a graph demonstrating DNA melting curves.

FIG. 13 c is a graph demonstrating the effects of temperature on therelative light absorbance of DNA.

FIG. 13 d is a graph demonstrating the effects of temperature on therelative optical absorbance of DNA.

FIG. 13 e is a graph demonstrating the effects of temperature on thefluorescence of DNA labeled with a pair of interactive labels.

FIG. 13 f is a graph demonstrating the effects of temperature on thefluorescence of DNA labeled with a pair of interactive labels.

FIG. 13 g is a graph demonstrating the effects of a target nucleic acidon the fluorescence of DNA labeled with a pair of interactive labels.

FIG. 14 demonstrates three templates (labeled 1, 2, and 3) that may beused to detect FEN nuclease activity.

FIG. 15 is a Sypro Orange stained polyacrylamide gel demonstratingCBP-tagged PFU FEN-1 protein.

FIG. 16 is an autoradiograph of a FEN-1 nuclease assay.

FIG. 17A-E illustrates one embodiment of the invention utilizing alabeled third oligonucleotide and template nucleic acid FIG. 17Aillustrates the formation of a first duplex.

FIG. 17B illustrates the polymerization or extension step of the firstduplex.

FIG. 17C illustrates the cleavage of the first duplex.

FIG. 17D illustrates the formation of the second duplex.

FIG. 17E illustrates the polymerization or extension step of the secondduplex.

FIG. 17F illustrates the cleavage and detection steps of the secondduplex.

FIG. 18 is a graphical representation of the data generated using oneembodiment of the invention in a real-time QPCR reaction.

FIG. 19A is a titration curve of the second oligonucleotide of theinvention in a Q-PCR reaction. The graph shows the fluorescent signalproduced upon cleavage of the first cleavage structure.

FIG. 19B is a titration curve of the second oligonucleotide of theinvention in a Q-PCR reaction. The graph shows the fluorescent signalproduced upon cleavage of the second cleavage structure.

FIG. 20A is a titration curve (100 nM to 600 nM) of the secondoligonucleotide of the invention in a Q-PCR reaction. The graph showsthe fluorescent signal produced upon cleavage of the first cleavagestructure.

FIG. 20B is a titration curve (100 nM to 600 nM) of the secondoligonucleotide of the invention in a Q-PCR reaction. The graph showsthe fluorescent signal produced upon cleavage of the second cleavagestructure.

FIG. 20C is a titration curve (700 nM to 900 nM) of the thirdoligonucleotide of the invention in a Q-PCR reaction. The graph showsthe fluorescent signal produced upon cleavage of the first cleavagestructure.

FIG. 21 is a representation of a key probe.

DESCRIPTION

The invention provides a method wherein, a target nucleic acid isdetected by sequentially forming and cleaving a first cleavage structurecomprising a target nucleic acid, and a first and secondoligonucleotide. The first cleavage structure is formed when the firstand second oligonucleotides hybridize to the target nucleic acid and thefirst oligonucleotide is extended by a polymerase. A 5′ flap of thesecond oligonucleotide is cleaved and released, serving as a primer in asecond cleavage structure. The second cleavage structure comprises atemplate nucleic acid, the released flap of the second oligonucleotideand a third oligonucleotide. The second cleavage structure is formedwhen the released flap and third oligonucleotide hybridize to thetemplate nucleic acid and the released flap is extended by a polymerase.The third oligonucleotide is cleaved which causes the disassociation ofthe third oligonucleotide from the template nucleic acid. The presenceand/or amount of the target nucleic acid is determined by detecting thecleavage and subsequent release of the third oligonucleotide from thetemplate nucleic acid.

The invention also provides a method wherein a target nucleic acid isdetected by linear amplification by sequentially forming and cleaving afirst cleavage structure comprising a target nucleic acid, and a firstand second oligonucleotide, wherein the cleavage reaction releases theflap of the second oligonucleotide; and forming and cleaving a secondcleavage structure comprising a template nucleic acid, the released flapof the second oligonucleotide and a third oligonucleotide, wherein thecleavage reaction releases the flap of the third oligonucleotide. Thepresence and/or amount of a target nucleic acid is determined bydetecting the released first and/or second flaps, wherein there is a 1:1ratio of released first flap to released second flap.

The invention also provides for an exponential amplification method fordetection of a target nucleic acid. A target nucleic acid is detected byexponential amplification by sequentially forming and cleaving a firstcleavage structure comprising a target nucleic acid, and a first andsecond oligonucleotide, wherein the cleavage reaction releases the flapof the second oligonucleotide; and forming and cleaving a secondcleavage structure comprising a template nucleic acid, the released flapof the second oligonucleotide, a third oligonucleotide, and a fourtholigonucleotide, wherein the cleavage reaction releases the flaps of thethird and fourth oligonucleotide. The presence and/or amount a targetnucleic acid is determined by detecting the released first and/or secondflaps, wherein there is a 1:2 ratio of released first flap to thecombination of released second flap and released third.

These methods of the invention are performed isothermally or underthermal cycling conditions.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiologyand recombinant DNA techniques, which are within the skill of the art.Such techniques are explained fully in the literature. See, e.g.,Sambrook, Fritsch & Maniatis, 1989, Molecular Cloning: A LaboratoryManual, Second Edition; Oligonucleotide Synthesis (M. J. Gait, ed.,1984); Nucleic Acid Hybridization (B. D. Harnes & S. J. Higgins, eds.,1984); A Practical Guide to Molecular Cloning (B. Perbal, 1984); and aseries, Methods in Enzymology (Academic Press, Inc.); Short Protocols InMolecular Biology, (Ausubel et al., ed., 1995). All patents, patentapplications, and publications mentioned herein, both supra and infra,are hereby incorporated by reference.

I. Nucleic Acids

A. Nucleic Acid Sequences Useful in the Invention

The invention provides for methods of detecting or measuring a targetnucleic acid sequence; and also utilizes oligonucleotides, primers andprobes for forming a cleavage structure according to the invention andprimers for amplifying a template nucleic acid sequence. As used herein,the terms “nucleic acid”, “polynucleotide” and “oligonucleotide” referto primers, probes, and oligomer fragments to be detected, and shall begeneric to polydeoxyribonucleotides (containing 2-deoxy-D-ribose), topolyribonucleotides (containing D-ribose), and to any other type ofpolynucleotide which is an N-glycoside of a purine or pyrimidine base,or modified purine or pyrimidine bases (including abasic sites) oranalogs such as protein nucleic acid (PNA). There is no intendeddistinction in length between the term “nucleic acid”, “polynucleotide”and “oligonucleotide”, and these terms will be used interchangeably.These terms refer only to the primary structure of the molecule. Thus,these terms include double- and single-stranded DNA, as well as double-and single-stranded RNA.

The complement of a nucleic acid sequence as used herein refers to anoligonucleotide which, when aligned with the nucleic acid sequence suchthat the 5′ end of one sequence is paired with the 3′ end of the other,is in “antiparallel association.”

The oligonucleotide is not necessarily physically derived from anyexisting or natural sequence but may be generated in any manner,including chemical synthesis, DNA replication, reverse transcription ora combination thereof. The terms “oligonucleotide” or “nucleic acid”intend a polynucleotide of genomic DNA or RNA, cDNA, semisynthetic, orsynthetic origin which, by virtue of its synthetic origin ormanipulation: (1) is not associated with all or a portion of thepolynucleotide with which it is associated in nature; and/or (2) islinked to a polynucleotide other than that to which it is linked innature.

Because mononucleotides are reacted to make oligonucleotides in a mannersuch that the 5′ phosphate of one mononucleotide pentose ring isattached to the 3′ oxygen of its neighbor in one direction via aphosphodiester linkage, an “end” or “terminus” of an oligonucleotide isreferred to as the “5′ end” if its 5′ phosphate is not linked to the 3′oxygen of a mononucleotide pentose ring and as the “3′ end” if its 3′oxygen is not linked to a 5′ phosphate of a subsequent mononucleotidepentose ring. As used herein, a nucleic acid sequence which is internalwithin a larger oligonucleotide, may be said to be a 5′ or a 3′ region,depending upon whether it is located closer to the 5′ or 3′ terminus ofthe molecule.

When two different, non-overlapping oligonucleotides anneal to differentregions of the same linear complementary nucleic acid sequence, and the3′ end of one oligonucleotide points toward the 5′ end of the other, theformer may be called the “upstream” oligonucleotide and the latter the“downstream” oligonucleotide.

Certain bases not commonly found in natural nucleic acids may beincluded in the nucleic acids of the present invention and include, forexample, inosine and 7-deazaguanine. Complementarity need not beperfect; stable duplexes may contain mismatched base pairs or unmatchedbases. Those skilled in the art of nucleic acid technology can determineduplex stability empirically considering a number of variablesincluding, for example, the length of the oligonucleotide, basecomposition and sequence of the oligonucleotide, ionic strength, andincidence of mismatched base pairs.

Stability of a nucleic acid duplex is measured by the meltingtemperature, or “T_(m)”. The T_(m) of a particular nucleic acid duplexunder specified conditions is the temperature at which half of the basepairs have disassociated.

B. Oligonucleotides Useful According to the Invention

The invention provides for oligonucleotide primers, oligonucleotideprobes, and template nucleic acids useful for detecting or measuring anucleic acid, for amplifying a template nucleic acid sequence, and forforming a cleavage structure according to the invention.

As used herein, “amplifying” refers to producing additional copies of anucleic acid sequence by isothermal methods or by methods that requirethermal cycling, including the method of the polymerase chain reaction.

1. Primers

The invention provides for a first oligonucleotide (defined herein) anda released flap of a second oligonucleotide (defined herein) that areextendible by polymerization.

The term “primer” may refer to more than one primer and refers to anoligonucleotide, whether occurring naturally, as in a purifiedrestriction digest, or produced synthetically, which is capable ofacting as a point of initiation of synthesis along a complementarystrand when placed under conditions in which synthesis of a primerextension product which is complementary to a nucleic acid strand iscatalyzed. Thus, a “primer” can include a first oligonucleotide (definedherein) and a released flap of a second oligonucleotide (definedherein). Conditions suitable for synthesis of a primer extension product(e.g., from a first oligonucleotide or from the released flap of asecond oligonucleotide, as defined herein) include the presence of fourdifferent deoxyribonucleoside triphosphates and apolymerization-inducing agent such as DNA polymerase or reversetranscriptase, in a suitable buffer (“buffer” includes substituentswhich are cofactors, or which affect pH, ionic strength, etc.), and at asuitable temperature. The primer is preferably single-stranded formaximum efficiency in amplification.

Oligonucleotide primers useful according to the invention aresingle-stranded DNA or RNA molecules that are hybridizable to a templatenucleic acid sequence and prime enzymatic synthesis of a second nucleicacid strand. The primer is complementary to a portion of a targetmolecule present in a pool of nucleic acid molecules. It is contemplatedthat oligonucleotide primers according to the invention are prepared bysynthetic methods, either chemical or enzymatic. Alternatively, such amolecule or a fragment thereof is naturally-occurring, and is isolatedfrom its natural source or purchased from a commercial supplier.

A “first oligonucleotide” according to the invention is preferably 6 to100, more preferably 8 to 30 and most preferably 20 nucleotides inlength. A “first” oligonucleotide is a least partially complementary tothe target nucleic acid.

A “released flap of a second oligonucleotide” according to the inventionis preferably 6-80 nucleotides, and most preferably about 10-25nucleotides. A “released flap of a second oligonucleotide” and a firstoligonucleotide are of a length that is sufficient to permithybridization to a target or template nucleic acid and extension bypolymerization.

Primers useful according to the invention are also designed to have aparticular melting temperature (Tm) by the method of melting temperatureestimation. Commercial programs, including Oligo™, Primer Design andprograms available on the internet, including Primer3 and OligoCalculator can be used to calculate a Tm of a nucleic acid sequenceuseful according to the invention. Preferably, the Tm of anamplification primer useful according to the invention, as calculatedfor example by Oligo Calculator, is preferably between about 15 and 80°C. and more preferably between about 50 and 60° C. Oligonucleotideprimers can be designed with these considerations in mind andsynthesized according to the following methods.

a. Oligonucleotide Primer Design Strategy

The design of a particular oligonucleotide primer according to theinvention, involves selecting a sequence that is capable of recognizingthe target sequence, but has a minimal predicted secondary structure.Furthermore, the Tm of the oligonucleotide is optimized by analysis ofthe length and GC content of the oligonucleotide. Furthermore, whendesigning a PCR primer useful for the amplification of genomic DNA, theselected primer sequence does not demonstrate significant matches tosequences in the GenBank database (or other available databases).

The design of a primer is facilitated by the use of readily availablecomputer programs, developed to assist in the evaluation of the severalparameters described above and the optimization of primer sequences.Examples of such programs are “PrimerSelect” of the DNAStar™ softwarepackage (DNAStar, Inc.; Madison, Wis.), OLIGO 4.0 (National Biosciences,Inc.), PRIMER, Oligonucleotide Selection Program, PGEN and AMPLIFY(described in Ausubel et al., 1995, Short Protocols in MolecularBiology, 3rd Edition, John Wiley & Sons). In one embodiment, primers aredesigned with sequences that serve as targets for other primers toproduce a PCR product that has known sequences on the ends which serveas targets for further amplification (e.g. to sequence the PCR product).If many different target nucleic acid sequences are amplified withspecific primers that share a common ‘tail’ sequence′, the PCR productsfrom these distinct genes can subsequently be sequenced with a singleset of primers. Alternatively, in order to facilitate subsequent cloningof amplified sequences, primers are designed with restriction enzymesite sequences appended to their 5′ ends. Thus, all nucleotides of theprimers are derived from a target nucleic acid sequence or sequencesadjacent to a target nucleic acid sequence, except for the fewnucleotides necessary to form a restriction enzyme site. Such enzymesand sites are well known in the art. If the genomic sequence of a targetnucleic acid sequence and the sequence of the open reading frame of atarget nucleic acid sequence are known, design of particular primers iswell within the skill of the art.

b. Synthesis

The primers themselves are synthesized using techniques that are alsowell known in the art. Methods for preparing oligonucleotides ofspecific sequence are known in the art, and include, for example,cloning and restriction digest analysis of appropriate sequences anddirect chemical synthesis. Once designed, oligonucleotides are preparedby a suitable chemical synthesis method, including, for example, thephosphotriester method described by Narang et al., 1979, Methods inEnzymology, 68:90, the phosphodiester method disclosed by Brown et al.,1979, Methods in Enzymology, 68:109, the diethylphosphoramidate methoddisclosed in Beaucage et al., 1981, Tetrahedron Letters, 22:1859, andthe solid support method disclosed in U.S. Pat. No. 4,458,066, or byother chemical methods using either a commercial automatedoligonucleotide synthesizer (which is commercially available) or VLSIPS™technology.

c. Labels

A primer according to the invention can be labeled (e.g., by theattachment of a radiolabel, a fluorescent label a quencher or any of thelabels recited in the section entitled “Cleavage Structure”. A labeledoligonucleotide primer is prepared according to methods well known inthe art (see Sambrook et al., supra; Ausubel et al., supra).

C. Probes

The invention provides for oligonucleotides probes useful for forming acleavage structure or a labeled cleavage structure as defined herein.Methods of preparing a labeled cleavage structure according to theinvention are provided in the section entitled “Cleavage Structure”below. The invention provides for second, third and fourtholigonucleotides (all defined hereinabove) that are components of one ormore of a first or second duplex, according to the invention, or a firstor second cleavage structure, according to the invention. As usedherein, “probe” refers to any one of a second, third or fourtholigonucleotide according to the invention.

A “second oligonucleotide” according to the invention is preferably20-120, more preferably 25-45 and most preferably 35 nucleotides inlength. A “second oligonucleotide” comprises a 3′ and a 5′ region. The3′ region of a “second oligonucleotide” is at least partiallycomplementary to the target nucleic acid. A 5′ region of a “secondoligonucleotide” is preferably 8 to 80 and most preferably 10 to 20nucleotides in length. In one embodiment of the invention, a 5′ regionof a “second oligonucleotide” is at least partially complementary to aregion of a target nucleic acid. In another embodiment of the invention,the 5′ region of the second oligonucleotide according to the inventionis not complementary to a target nucleic acid.

A “third oligonucleotide” and “fourth oligonucleotide” according to theinvention are preferably 20-120, more preferably 25-45 and mostpreferably 35 nucleotides in length. A “third oligonucleotide” and“fourth oligonucleotide” comprise a 3′ and a 5′ region. Third and fourtholigonucleotides according to the invention comprise a 3′ region that isat least partially complementary to a region of a template nucleic acidand is preferably 8 to 80 and most preferably 10-20 nucleotides. A 5′region of a “third oligonucleotide” or “fourth oligonucleotide” ispreferably 8 to 80 and most preferably 10 to 20 nucleotides in length.In an embodiment of the invention, a 5′ region of a “thirdoligonucleotide” or “fourth oligonucleotide” is at least partiallycomplementary to a region of a template nucleic acid. In anotherembodiment of the invention, the 5′ region of the “thirdoligonucleotide” or “fourth oligonucleotide” according to the inventionis not complementary to a template nucleic acid.

The probe, preferably, does not contain a sequence complementary tosequence(s) used in the primer extension (s). Generally the 3′ terminusof the probe will be “blocked” to prohibit incorporation of the probeinto a primer extension product.

1. Labels

A probe according to the invention can be labeled (e.g., by theattachment of a radiolabel, a fluorescent label a quencher or any of thelabels recited in the section entitled “Cleavage Structure”). A labeledoligonucleotide probe is prepared according to methods well known in theart (see Sambrook et al., supra; Ausubel et al., supra).

2. Probes Comprising a Secondary Structure

A “probe” according to one embodiment of the invention can be a singlestranded nucleic acid comprising a region or regions that arecomplementary to a target or a template nucleic acid sequence (e.g.,target or template nucleic acid binding sequences) (for example c inFIG. 7). A “probe” according to this embodiment of the invention has asecondary structure that changes upon binding of the probe to the targetor template nucleic acid sequence and can further comprise a bindingmoiety. A “probe” according to this embodiment of the invention binds toa target or template nucleic acid sequence to form a cleavage structurethat can be cleaved by a cleavage means, wherein cleaving is performedat a cleaving temperature, and wherein the secondary structure of theprobe when not bound to the target or template nucleic acid sequence ispreferably stable at or below the cleaving temperature. A probeaccording to the invention cannot be cleaved to generate a signal by a“cleavage means”, as defined herein, prior to binding to a target ortemplate nucleic acid. In one embodiment of the invention, a probe maycomprise a region that cannot bind or is not complementary to a targetor template nucleic acid sequence. In another embodiment of theinvention, a probe does not have a secondary structure when bound to atarget or template nucleic acid.

As used herein, “secondary structure” refers to a three-dimensionalconformation (for example a hairpin, a stem-loop structure, an internalloop, a bulge loop, a branched structure or a pseudoknot, FIGS. 8, 9 and21; multiple stem loop structures, cloverleaf type structures or anythree dimensional structure. As used herein, “secondary structure”includes tertiary, quaternary etc. . . . structure. A probe comprisingsuch a three-dimensional structure binds to a target or template nucleicacid sequence to form a cleavage structure that can be cleaved by acleavage means at a cleaving temperature. The three dimensionalstructure of the probe when not bound to the target or template nucleicacid sequence is preferably stable at or below the cleaving temperature.“Secondary structure” as used herein, means a sequence comprising afirst single-stranded sequence of bases (referred to herein as a“complementary nucleic acid sequence” (for example b′ in FIG. 7))followed by a second complementary sequence either in the same molecule(for example b′ in FIG. 7), or in a second molecule comprising theprobe, folds back on itself to generate an antiparallel duplexstructure, wherein the single-stranded sequence and the complementarysequence (that is, the complementary nucleic acid sequences) anneal bythe formation of hydrogen bonds. Oligonucleotide probes, as used in thepresent invention include oligonucleotides comprising secondarystructure, including, but not limited to molecular beacons, safety pins(FIG. 10), scorpions (FIG. 11), sunrise/amplifluor probes (FIG. 12), andkey probes (FIG. 21) the details and structures of which are describedbelow and in the corresponding figures.

As used herein, first and second “complementary” nucleic acid sequencesare complementary to each other and can anneal by the formation ofhydrogen bonds between the complementary bases.

A secondary structure also refers to the conformation of a nucleic acidmolecule comprising an affinity pair, defined herein, wherein theaffinity pair reversibly associates as a result of attractive forcesthat exist between the pair of moieties comprising the affinity pair. Asused herein, if a probe according to this embodiment further comprises abinding moiety, secondary structure prevents the binding moiety on theprobe from binding to a capture element, and a change in secondarystructure upon binding of the probe to the target nucleic acid andsubsequent cleavage of the bound probe permits the binding moiety to becaptured by the capture element.

A “probe” according to this embodiment of the invention can beunimolecular. As used herein, a “unimolecular” probe comprises a singlemolecule that binds to a target or template nucleic acid sequence toform a cleavage structure that can be cleaved by a cleavage means,wherein cleaving is performed at a cleaving temperature, and wherein thesecondary structure of the “unimolecular” probe when not bound to thetarget or template nucleic acid sequence is preferably stable at orbelow the cleaving temperature. Unimolecular probes useful according tothe invention include but are not limited to beacon probes, probescomprising a hairpin, stem-loop, internal loop, bulge loop or pseudoknotstructure, scorpion probes and sunrise/amplifluor probes.

A “probe” according to this embodiment of the invention can be more thanone molecule (e.g., bi-molecular or multi-molecular). At least one ofthe molecules comprising a bi-molecular or multi-molecular probe bindsto a target or template nucleic acid sequence to form a cleavagestructure that can be cleaved by a cleavage means, wherein cleaving isperformed at a cleaving temperature, and wherein the secondary structureof the molecule of the probe when not bound to the target or templatenucleic acid sequence is preferably stable at or below the cleavingtemperature. The molecules comprising the multimolecular probe associatewith each other via intermolecular bonds (e.g., hydrogen bonds orcovalent bonds). For example, a heterologous loop (see FIG. 8, FIG. 21),or a cloverleaf structure wherein one or more loops of the cloverleafstructure (see FIG. 9) comprises a distinct molecule, and wherein themolecules that associate to form the cloverleaf structure associate viaintermolecular bonding (e.g., hydrogen bonding or covalent bonding), areexamples of multimolecular probes useful according to the invention.

As used herein, a “molecule” refers to a polynucleotide, and includes apolynucleotide further comprising an attached member or members of anaffinity pair.

A “probe” or a “molecule” comprising a probe, according to thisembodiment of the invention, is 5-10,000 nucleotides in length, ideallyfrom 17-40 nucleotides in length, although probes or a moleculecomprising a probe of different lengths are useful.

A “probe” according to this embodiment of the invention has a target ortemplate nucleic acid binding sequence that is from about 5 to about10,000 nucleotides, and preferably from 10 to about 140 nucleotides. Inone embodiment of the invention, a “probe” according to the inventioncomprises at least first and second complementary nucleic acid sequencesor regions that are 3-250, preferably 4-15, and more preferably 5-11nucleotides long. The first and second complementary nucleic acidsequences may or may not have the same length. The invention providesfor a probe wherein the first and second complementary nucleic acidsequences are both located upstream (5′) of the target or templatenucleic acid binding site. Alternatively, the first and secondcomplementary nucleic acid sequences can both be located downstream (3′)of the target or template nucleic acid binding site. In anotherembodiment, the invention provides for a probe wherein the firstcomplementary nucleic acid sequence is upstream (5′) of the target ortemplate nucleic acid binding site and the second complementary nucleicacid sequence is downstream (3′) of the target or template nucleic acidbinding site. In another embodiment, the invention provides for a probewherein the second complementary nucleic acid sequence is upstream (5′)of the target or template nucleic acid binding site and the firstcomplementary nucleic acid sequence is downstream (3′) of the target ortemplate nucleic acid binding site. The actual length will be chosenwith reference to the target or template nucleic acid binding sequencesuch that the secondary structure of the probe is stable when the probeis not bound to the target or template nucleic acid at the temperatureat which cleavage of a cleavage structure comprising the probe bound toa target or template nucleic acid is performed. As the target ortemplate nucleic acid binding sequence increases in size up to 500nucleotides, the length of the complementary nucleic acid sequences mayincrease up to 15-125 nucleotides. For a target or template nucleic acidbinding sequence greater than 100 nucleotides, the length of thecomplementary nucleic acid sequences are not increased further. If theprobe is also an allele-discriminating probe, the lengths of thecomplementary nucleic acid sequences are more restricted, as isdiscussed below.

As used herein, the “target nucleic acid binding sequence” refers to theregion of the probe that binds specifically to the target nucleic acid.

As used herein, the “template nucleic acid binding sequence” refers tothe region of the probe that binds specifically to the template nucleicacid.

A probe according to the invention is capable of forming a secondarystructure, as defined herein, (including a stem loop, a hairpin, aninternal loop, a bulge loop, a branched structure and a pseudoknot) ormultiple secondary structures or any three-dimensional structure asdefined herein.

For example, according to one embodiment of the present invention, aprobe can be an oligonucleotide with secondary structure such as ahairpin or a stem-loop, and includes, but is not limited to molecularbeacons, safety pins, scorpions, sunrise/amplifluor probes, and keyprobes.

Molecular beacon probes comprise a hairpin, or stem-loop structure whichpossesses a pair of interactive signal generating labeled moieties(e.g., a fluorophore and a quencher) effectively positioned to quenchthe generation of a detectable signal when the beacon probe is nothybridized to the target nucleic acid sequence. The loop comprises aregion that is complementary to a target nucleic acid. The loop isflanked by 5′ and 3′ regions (“arms”) that reversibly interact with oneanother by means of complementary nucleic acid sequences when the regionof the probe that is complementary to a nucleic acid target sequence isnot bound to the target nucleic acid. Alternatively, the loop is flankedby 5′ and 3′ regions (“arms”) that reversibly interact with one anotherby means of attached members of an affinity pair to form a secondarystructure when the region of the probe that is complementary to anucleic acid target sequence is not bound to the target nucleic acid. Asused herein, “arms” refers to regions of a molecular beacon probe thata) reversibly interact with one another by means of complementarynucleic acid sequences when the region of the probe that iscomplementary to a nucleic acid target sequence is not bound to thetarget nucleic acid or b) regions of a probe that reversibly interactwith one another by means of attached members of an affinity pair toform a secondary structure when the region of the probe that iscomplementary to a nucleic acid target sequence is not bound to thetarget nucleic acid. When a molecular beacon probe is not hybridized totarget, the arms hybridize with one another to form a stem hybrid, whichis sometimes referred to as the “stem duplex”. This is the closedconformation. When a molecular beacon probe hybridizes to its target the“arms” of the probe are separated. This is the open conformation. In theopen conformation an arm may also hybridize to the target. Such probesmay be free in solution, or they may be tethered to a solid surface.When the arms are hybridized (e.g., form a stem) the quencher is veryclose to the fluorophore and effectively quenches or suppresses itsfluorescence, rendering the probe dark. Such probes are described inU.S. Pat. No. 5,925,517 and U.S. Pat. No. 6,037,130.

As used herein, a molecular beacon probe can also be an“allele-discriminating” probe as described herein.

Molecular beacon probes have a fluorophore attached to one arm and aquencher attached to the other arm. The fluorophore and quencher, forexample, tetramethylrhodamine and DABCYL, need not be a FRET pair.

For stem loop probes useful in this invention, the length of the probesequence that is complementary to the target, the length of the regionsof a probe (e.g., stem hybrid) that reversibly interact with one anotherby means of complementary nucleic acid sequences, when the regioncomplementary to a nucleic acid target sequence is not bound to thetarget nucleic acid, and the relation of the two, is designed accordingto the assay conditions for which the probe is to be utilized. Thelengths of the target-complementary sequences and the stem hybridsequences for particular assay conditions can be estimated according towhat is known in the art. The regions of a probe that reversiblyinteract with one another by means of complementary nucleic acidsequences when the region of the probe that is complementary to anucleic acid target sequence is not bound to the target nucleic acid arein the range of 6 to 100, preferably 8 to 50 nucleotides and mostpreferably 8 to 25 nucleotides each. The length of the probe sequencethat is complementary to the target is preferably 17-40 nucleotides,more preferably 17-30 nucleotides and most preferably 17-25 nucleotideslong.

The oligonucleotide sequences of molecular beacon probes modifiedaccording to this invention may be DNA, RNA, cDNA or combinationsthereof. Modified nucleotides may be included, for examplenitropyrole-based nucleotides or 2′-O-methylribonucleotides. Modifiedlinkages also may be included, for example phosphorothioates. Modifiednucleotides and modified linkages may also be incorporated inwavelength-shifting primers according to this invention.

A safety pin probe, as utilized in the present invention, requires a“universal” hairpin probe 1 (FIG. 10, b171), comprising a hairpinstructure, with a fluorophore (FAM) on the 5′ arm of the hairpin and aquencher (Dabcyl) on the 3′ arm, and a probe 2 (FIG. 10, SP170a)comprising a stem-loop comprising two domains: the 5′ two thirds ofprobe 2 have a (universal) sequence complementary to the hairpin probe1, and nucleotides that will stop the DNA polymerase, and the 3′ onethird of probe 2, which serves as the target specific primer. As thepolymerase, primed from the reverse primer (that is, the 3′ one third ofprobe 2) synthesizes the top strand, the 5′ end of probe 2 will bedisplaced and degraded by the 5′ exonucleolytic activity until the “stopnucleotides” are reached. At this time the remainder of probe 2 opens upor unfolds and serves as a target for hairpin probe 1, therebyseparating the fluorophore from the quencher (FIG. 10).

Scorpion probes, as used in the present invention comprise a 3′ primerwith a 5′ extended probe tail comprising a hairpin structure whichpossesses a fluorophore/quencher pair. The probe tail is “protected”from replication in the 5′→3′ direction by the inclusion of hexethlyeneglycol (HEG) which blocks the polymerase from replicating the probe.During the first round of amplification the 3′ target-specific primeranneals to the target and is extended such that the scorpion is nowincorporated into the newly synthesized strand, which possesses a newlysynthesized target region for the 5′ probe. During the next round ofdenaturation and annealing, the probe region of the scorpion hairpinloop will hybridize to the target, thus separating the fluorophore andquencher and creating a measurable signal. Such probes are described inWhitcombe et al., Nature Biotechnology 17: 804-807 (1999), and in FIG.11.

An additional oligonucleotide probe useful in the present invention isthe sunrise/amplifluor probe. The sunrise/amplifluor probe is of similarconstruction as the scorpion probe with the exception that is lacks theHEG monomer to block the 5′→3′ replication of the hairpin probe region.Thus, in the first round of amplification, the 3′ target specific primerof the sunrise/amplifluor anneals to the target and is extended, thusincorporating the hairpin probe into the newly synthesized strand(sunrise strand). During the second round of amplification a second,non-labeled primer anneals to the 3′ end of the sunrise strand (Cycle 2in FIG. 12). However, as the polymerase reaches the 5′ end of thehairpin, due to the lack of the HEG stop sequence, the polymerase willdisplace and replicate the hairpin, thus separating the fluorophore andquencher, and incorporating the linearized hairpin probe into the newstrand. Probes of this type are described further in Nazarneko et al.,Nucleic Acid Res. 25: 2516-2521 (1997), and in FIG. 12.

An additional oligonucleotide probe useful in the present invention isthe key probe. The key probe is described in U.S. Application No.60/665,400, which is herein incorporated by reference in its entirety.The key probe can be formed from a target binding oligonucleotide joinedto its complementary sequence, optionally through a linkeroligonucleotide sequence, to form a hairpin structure comprising stemand, optionally, loop portions (See FIG. 21). The loop portion containsthe optional linker sequence, or just a covalent bond joining the endsof the first and second sequences. The linker sequence, which forms theloop structure, may or may not hybridize to the target sequence. No morethan 5 contiguous nucleotides of the optional linker sequence arecomplementary to the target sequence. The stem portion optionallycontains one or more mismatches between the first and second sequences;the number of mismatches can be used to adjust the melting temperatureof the probe. The stem portion may also contain modified nucleotides,for example minor groove binders or LNA, which can alter the affinity ofthe probe for the target sequence and also can be used to adjust themelting temperature. The key probe may also contain an interactive pairof labels, for example a fluorophore/quencher pair, attached at or nearthe ends of the probe such that a detectable signal, e.g., increasedfluorescence emission of the fluorophore, is produced when the probehybridizes to the target sequence.

For safety pin, scorpion, sunrise/amplifluor, and key probes useful inthis invention, the length of the probe sequence that is complementaryto the target, the length of the regions of a probe (e.g., stem hybrid)that reversibly interact with one another by means of complementarynucleic acid sequences when the region complementary to a nucleic acidtarget sequence is not bound to the target nucleic acid and the relationof the two is designed according to the assay conditions for which theprobe is to be utilized. The lengths of the target-complementarysequences and the stem hybrid sequences for particular assay conditionscan be estimated according to what is known in the art. The regions of aprobe that reversibly interact with one another by means ofcomplementary nucleic acid sequences when the region complementary to anucleic acid target sequence is not bound to the target nucleic acid arein the range of 6 to 100, preferably 8 to 50 nucleotides and mostpreferably 8 to 25 nucleotides each. The length of the probe sequencethat is complementary to the target is preferably 17-40 nucleotides,more preferably 17-30 nucleotides and most preferably 17-25 nucleotideslong. The stability of the interaction between the regions of a probethat reversibly interact with one another by means of complementarynucleic acid sequences is determined by routine experimentation toachieve proper functioning. In addition to length, the stability of theinteraction between the regions of a probe that reversibly interact withone another by means of complementary nucleic acid sequences between theregions of a probe that reversibly interact with one another by means ofcomplementary nucleic acid sequences can be adjusted by altering the G-Ccontent and inserting destabilizing mismatches. One of the regions of aprobe that reversibly interact with one another by means ofcomplementary nucleic acid sequences can be designed to be partially orcompletely complementary to the target. If the 3′ arm is complementaryto the target the probe can serve as a primer for a DNA polymerase.Also, wavelength-shifting molecular beacon probes can be immobilized tosolid surfaces, as by tethering, or be free-floating.

A wide range of fluorophores may be used in probes and primers accordingto this invention. Available fluorophores include coumarin, fluorescein,tetrachlorofluorescein, hexachlorofluorescein, Lucifer yellow,rhodamine, BODIPY, tetramethylrhodamine, Cy3, Cy5, Cy7, eosine, Texasred and ROX. Combination fluorophores such as fluorescein-rhodaminedimers, described, for example, by Lee et al. (1997), Nucleic AcidsResearch 25:2816, are also suitable. Fluorophores may be chosen toabsorb and emit in the visible spectrum or outside the visible spectrum,such as in the ultraviolet or infrared ranges.

Suitable quenchers described in the art include particularly DABCYL andvariants thereof, such as DABSYL, DABMI and Methyl Red. Fluorophores canalso be used as quenchers, because they tend to quench fluorescence whentouching certain other fluorophores. Preferred quenchers are eitherchromophores such as DABCYL or malachite green, or fluorophores that donot fluoresce in the detection range when the probe is in the openconformation.

A “hairpin structure” or a “stem” refers to a double-helical regionformed by base pairing between adjacent, inverted, complementarysequences in a single strand of RNA or DNA.

A “stem-loop” structure refers to a hairpin structure, furthercomprising a loop of unpaired bases at one end.

As used herein, a probe with “stable” secondary structure when not boundto a target or template nucleic acid sequence, refers to a secondarystructure wherein 50% or more (e.g., 50%, 55%, 75% or 100%) of the basepairs that constitute the probe are not dissociated under conditionswhich permit hybridization of the probe to the target or templatenucleic acid, but in the absence of the target or template nucleic acid.

“Stability” of a nucleic acid duplex is determined by the meltingtemperature, or “T_(m)”. The T_(m) of a particular nucleic acid duplexunder specified conditions (e.g., salt concentration and/or the presenceor absence of organic solvents) is the temperature at which half (50%)of the base pairs of the duplex molecule have disassociated (that is,are not hybridized to each other in a base-pair).

The “stability” of the secondary structure of a probe when not bound tothe target or template nucleic acid is defined in a melting temperatureassay, in a fluorescence resonance energy transfer (FRET) assay or in afluorescence quenching assay, (the details or which are described in asection entitled, “Determining the Stability or the Secondary Structureof a Probe”).

A probe useful according to this embodiment of the invention must havesecondary structure that is “stable” at or below the temperature of thecleavage reaction. Thus, the temperature at which cleavage of a cleavagestructure by a cleavage means is performed according to the invention,must be lower than the Tm of the secondary structure of the probe. Thesecondary structure of the probe is “stable” in a melting temperatureassay at a temperature that is at or below the temperature of thecleavage reaction (i.e., at which cleavage is performed) if the level oflight absorbance at the temperature at or below the temperature of thecleavage reaction is less than (i.e., at least 5% less than, preferably20% less than and most preferably 25% less than etc. . . .) than thelevel of light absorbance at a temperature that is equal to the Tm ofthe probe.

According to the method of the invention, the stability of a secondarystructure is measured by a FRET assay or a fluorescence quenching assay(described in the section entitled, “Determining the Stability of theSecondary Structure of a Probe”). As used herein, a fluorescencequenching assay can include a FRET assay. A probe according to theinvention is labeled with an appropriate pair of interactive labels(e.g., a FRET pair (for example as described in the section entitled,“Determining the Stability of the Secondary Structure of the Probe”,below) that can interact over a distance of, for example 2 nucleotides,or a non-FRET-pair, (e.g., tetramethylrhodamine and DABCYL) that caninteract over a distance of, for example, 20 nucleotides. For example, aprobe according to this embodiment of the invention may be labeled witha fluorophore and a quencher and fluorescence is then measured, in theabsence of a target nucleic acid, at different temperatures. The Tm isthe temperature at which the level of fluorescence is 50% of the maximallevel of fluorescence observed for a particular probe, see FIG. 13 e.The Tm for a particular probe wherein the nucleic acid sequence of theprobe is known, can be predicted according to methods known in the art.Thus, fluorescence is measured over a range of temperatures, e.g.,wherein the lower temperature limit of the range is at least 50° below,and the upper temperature limit of the range is at least 50° above theTm or predicted Tm, for a probe according to the invention.

A secondary structure is herein defined as “stable” in a FRET assay at atemperature that is at or below the cleaving temperature if the level offluorescence is increased or decreased (e.g., at least 5% less than,preferably 20% less than and more preferably 25 less than, etc. . . .)the level of FRET that is observed at the Tm of the probe (see FIGS. 13e and f). For example, an increase or a decrease in FRET car occur in aFRET assay according to the invention. In another embodiment, a shift inwavelength, which results in an increase in the new, shifted wavelengthor, a decrease in the new shifted wavelength, can occur in a FRET assayaccording to the invention.

A “change” in a secondary structure, according to the invention ismeasured in a FRET assay wherein a probe according to this embodiment ofthe invention comprises a fluorophore and a quencher that are positionedsuch that in the absence of a target or template nucleic acid, and attemperatures below the Tm of the probe there is quenching of the FRETpair (as described above). As used herein, a “change” in secondarystructure that occurs when a probe according to the invention binds to atarget nucleic acid, refers to an increase in fluorescence in such anassay, such that the level of fluorescence after binding of the probe tothe target or template nucleic acid at a temperature below the Tm of theprobe, is greater than (e.g., at least 5%, preferably 5-20% and mostpreferably 25 or more) the level of fluorescence observed in the absenceof a target or template nucleic acid sequence (see FIG. 13 g).

A secondary structure, according to this embodiment of the invention, isdetected by subjecting a probe comprising a fluorophore and a quencherto a FRET assay (as described above). A probe according to thisembodiment of the invention that exhibits a change in fluorescence thatcorrelates with a change in temperature (e.g., fluorescence increases asthe temperature of the FRET reaction is increased) is capable of forminga secondary structure.

As used herein, a “cleaving temperature” that is useful according tothis embodiment of the invention is a temperature that is less than (atleast 1° C. and preferably 10° C.) the T_(m) of a probe having asecondary structure. The “cleaving temperature” is initially selected tobe possible and preferably optimal for the particular cleavage meansbeing employed in the cleavage reaction.

Generally the 3′ terminus of the probe will be “blocked” to prohibitincorporation of the probe into a primer extension product if an activepolymerase is used in the reaction. “Blocking” can be achieved by usingnon-complementary bases or by adding a chemical moiety such as biotin ora phosphate group to the 3′ hydroxyl of the last nucleotide, which may,depending upon the selected moiety, serve a dual purpose by also actingas a label for subsequent detection or capture of the nucleic acidattached to the label. Blocking can also be achieved by removing the3′-OH or by using a nucleotide that lacks a 3′-OH such asdideoxynucleotide.

The term probe encompasses an allele-discriminating probe. As usedherein, an “allele-discriminating” probe preferentially hybridizes toperfectly complementary target nucleic acid sequences and discriminatesagainst sequences that vary by at least one nucleotide. A nucleic acidsequence which differs by at least one nucleotide, as compared to atarget nucleic acid sequence, hereafter referred to as a “target-likenucleic acid sequence”, is thus not a target nucleic acid sequence foran allele-discriminating probe according to the invention.

Allele-discriminating probes do not hybridize sufficiently to atarget-like nucleic acid sequence that contains one or more nucleotidemismatches as compared to the target nucleic acid complementarysequence, at a particular temperature or within a range of temperaturesdetermined by experimental optimization according to methods well knownin the art, and thus do not undergo a change in secondary structure uponbinding to a target-like nucleic acid sequence in the presence of only atarget-like nucleic acid sequence, and under conditions that wouldsupport hybridization of the allele discriminating probe to a targetnucleic acid sequence.

In one embodiment, an “allele-discriminating probe” according to theinvention refers to a probe that hybridizes to a target-like nucleicacid sequence that varies by at least one nucleotide from the targetnucleic acid sequence, wherein the variant nucleotide(s) is/are notlocated in the allele-discriminating site. According to this embodimentof the invention, “an allele-discriminating probe” cannot bind to atarget-like nucleic acid sequence that also varies by at least onenucleotide in the allele-discriminating site at a particular temperatureor within a range of temperatures determined by experimentaloptimization according to methods well known in the art. Singlenucleotide differences only affect the percentage of a probe that isbound to a target or target-like nucleic acid sequence. For example, theinvention provides for a perfectly matched probe, wherein as much as100% of the target or template is in a probe-target or probe-templatecomplex (e.g., is bound by probe), in the presence of excess probe. Theinvention also provides for probes comprising at least a single basemismatch wherein at least 1-5% and preferably 5-10% of the target-likeor template-like sequence is bound by the probe under the sameconditions used to form a complex comprising a target or templatesequence and a perfectly matched probe.

As used herein, “allele-discriminating site” refers to a region of atarget nucleic acid sequence that is different (i.e., by at least onenucleotide) from the corresponding region in all possible allelescomprising the target nucleic acid sequence.

Allele-discriminating probes useful according to the invention alsoinclude probes that bind less effectively to a target-like sequence, ascompared to a target sequence. The effectiveness of binding of a probeto a target sequence or a target-like sequence is measured in a FRETassay, performed at a temperature that is below (at least 5° C. andpreferably 10° C. or more) the Tm of the secondary structure of probe,in the presence of a target-like sequence or a target sequence. Thechange in the level of fluorescence in the presence or absence of atarget sequence compared to the change in the level of fluorescence inthe presence or absence of a target-like sequence, provides an effectivemeasure of the effectiveness of binding of a probe to a target ortarget-like sequence.

In a method according to the invention, a probe that binds lesseffectively to a target-like sequence as compared to a target sequencewould undergo a smaller (e.g., preferably 25-50%, more preferably 50-75%and most preferably 75-90% of the value of the change in fluorescenceupon binding to a target nucleic acid sequence) change in secondarystructure, as determined by measuring fluorescence in a FRET assay asdescribed herein, upon hybridization to a target-like sequence ascompared to a target nucleic acid sequence. In a method according to theinvention, a probe that binds less effectively to a target-like sequenceas compared to a target sequence would generate a signal that isindicative of the presence of a target-like nucleic acid sequence in asample. However, the intensity of the signal would be altered (e.g.,preferably 25-50%, more preferably 50-75% and most preferably 75-90%less than or more than the value of the change in fluorescence uponbinding to a target nucleic acid sequence) the intensity of a signalgenerated in the presence of a target sequence, as described hereinabovefor a smaller change.

A “signal that is indicative of the presence of a target nucleic acidsequence” or a “target-like nucleic acid sequence” refers to a signalthat is equal to a signal generated from 1 molecule to 10²⁰ molecules,more preferably about 100 molecules to 10¹⁷ molecules and mostpreferably about 1000 molecules to 10¹⁴ molecules of a target nucleicacid sequence or a target-like nucleic acid sequence.

3. Probes Comprising Secondary Structure and a Binding Moiety

A probe according to one embodiment of the invention can have asecondary structure that changes upon binding of the probe to the targetnucleic acid sequence and further comprises a binding moiety.

As used herein, a “binding moiety” refers to a region of a probe (forexample ab in FIG. 7) that is released upon cleavage of the probe by acleavage means and binds specifically to a capture element as a resultof attractive forces that exist between the binding moiety and thecapture element, and wherein specific binding between the binding moietyand the capture element only occurs when the secondary structure of theprobe has “changed”, as defined herein. “Binds specifically” means viahydrogen binding with a complementary nucleic acid or via an interactionbetween for example, the binding moiety and a binding protein capable ofbinding specifically to the nucleic acid sequence of the binding moiety.A “binding moiety” does not interfere with the ability of a probe tobind to a target or template nucleic acid sequence. A binding moiety isincapable of binding to a capture element when the probe is in itsnative secondary structural conformation and that, upon binding to atarget or template nucleic acid, the secondary structure changes in away that allows the binding moiety to bind to the capture element,preferably after cleavage by a cleavage agent. In one embodiment, theregion of a probe that is cleaved to form a binding moiety cannothybridize to a target nucleic acid sequence but can bind to a templatenucleic acid. The region of a “binding moiety” that is not a“complementary nucleic acid sequence”, as defined herein, (e.g., a inFIG. 7), is from 1-60 nucleotides, preferably from 1-25 nucleotides andmost preferably from 1-10 nucleotides in length. Methods of detectingspecific binding between a binding moiety as defined herein, and acapture element as defined herein, are well known in the art (see forexample, Sambrook et al., supra; Ausubel et al., supra).

In one embodiment of the invention, a probe, according to thisembodiment of the invention, further comprises a “reporter”.

As used herein, a “reporter” refers to a “label”, defined hereinbelowand/or a “tag” defined herein.

As used herein, “label” or “labeled moiety capable of providing asignal” refers to any atom or molecule which can be used to provide adetectable (preferably quantifiable) signal, and which can beoperatively linked to a nucleic acid. Labels may provide signalsdetectable by fluorescence, radioactivity, colorimetry, gravimetry,X-ray diffraction or absorption, magnetism, enzymatic activity, massspectrometry, binding affinity, hybridization radiofrequency,nanocrystals and the like. A labeled probe according to this embodimentof the invention is labeled at the 5′ end, the 3′ end or internally. Thelabel can be “direct”, i.e. a dye, or “indirect”. i.e. biotin, digoxin,alkaline phosphatase (AP), horse radish peroxidase (HRP) etc. . . . Fordetection of “indirect labels” it is necessary to add additionalcomponents such as labeled antibodies, or enzyme substrates to visualizethe, captured, released, labeled nucleic acid fragment. In oneembodiment of the invention, a label cannot provide a detectable signalunless the secondary structure has “changed”, as defined herein, (forexample, such that the binding moiety is accessible).

As used herein, a “binding moiety” also refers to a tag. As used herein,a “tag” refers to a moiety that is operatively linked to the 5′ end of aprobe (for example R in FIG. 7) and specifically binds to a captureelement as a result of attractive forces that exist between the tag andthe capture element, and wherein specific binding between the tag andthe capture element only occurs when the secondary structure of theprobe has changed (for example, such that the tag is accessible to acapture element). “Specifically binds” as it refers to a “tag” and acapture element means via covalent or hydrogen bonding or electrostaticattraction or via an interaction between, for example a protein and aligand, an antibody and an antigen, protein subunits, or a nucleic acidbinding protein and a nucleic acid binding site. A tag does notinterfere with the ability of a probe to anneal to a target or templatenucleic acid sequence. Second binding moieties include but are notlimited to biotin, streptavidin, a hapten, a protein, or a chemicallyreactive moiety.

As used herein, a “capture element” refers to a substance that isirreversibly attached to a solid substrate for example by chemicalcrosslinking or covalent binding, wherein the substance specificallybinds to (e.g., via hydrogen bonding or via an interaction between, anucleic acid binding protein and a nucleic acid binding site or betweencomplementary nucleic acids) a binding moiety as a result of attractiveforces that exist between the binding moiety and the capture element,and wherein specific binding between the binding moiety and the captureelement only occurs when the secondary structure of the probe comprisingthe binding moiety has “changed”, as defined herein. Capture elementsinclude but are not limited to a nucleic acid binding protein or anucleotide sequence.

As used herein, a “capture element” also refers to a substance that isirreversibly attached to a solid substrate for example by chemicalcrosslinking or covalent binding, wherein the substance specificallybinds to (e.g. via covalent or hydrogen bonding or electrostaticattraction via an interaction between, for example a protein and aligand, an antibody and an antigen, protein subunits, a nucleic acidbinding protein and a nucleic acid binding site or between complementarynucleic acids) a tag as a result of attractive forces that exist betweenthe tag and the capture element, and wherein specific binding betweenthe tag and the capture element only occurs when the secondary structureof the probe comprising the binding moiety has “changed”, as definedherein. Capture elements include but are not limited to biotin,streptavidin, a hapten, a protein, or a chemically reactive moiety.

As used herein, “solid support” means a surface to which a molecule(e.g. a first or capture element) can be irreversibly bound, includingbut not limited to membranes, sepharose beads, magnetic beads, tissueculture plates, silica based matrices, membrane based matrices, beadscomprising surfaces including but not limited to styrene, latex orsilica based materials and other polymers for example cellulose acetate,teflon, polyvinylidene difluoride, nylon, nitrocellulose, polyester,carbonate, polysulphone, metals, zeolites, paper, alumina, glass,polypropyle, polyvinyl chloride, polyvinylidene chloride,polytetrafluorethylene, polyethylene, polyamides, plastic, filter paper,dextran, germanium, silicon, (poly)tetrafluorethylene, gallium arsenide,gallium phosphide, silicon oxide, silicon nitrate and combinationsthereof. Methods of attaching a capture element as defined herein arewell known in the art and are defined hereinbelow. Additional solidsupports are also discussed below.

As used herein, “affinity pair” refers to a pair of moieties (forexample complementary nucleic acid sequences, protein-ligand,antibody-antigen, protein subunits, and nucleic acid bindingproteins-binding sites) that can reversibly associate as a result ofattractive forces that exist between the moieties. An “affinity pair”includes the combination of a binding moiety and the correspondingcapture element and the combination of a tag and the correspondingcapture element.

In embodiments wherein the affinity pair comprises complementary nucleicacid regions that reversibly interact with one another, the lengths ofthe target nucleic acid binding sequences, and the nucleic acidsequences comprising the affinity pair, are chosen for the properthermodynamic functioning of the probe under the conditions of theprojected hybridization assay. Persons skilled in hybridization assayswill understand that pertinent conditions include probe, target andsolute concentrations, detection temperature, the presence ofdenaturants and volume excluders, and other hybridization-influencingfactors. The length of a target or template nucleic acid bindingsequence can range from 7 to about 10,000 nucleotides, preferably from10 nucleotides to about 140 nucleotides. If the probe is also anallele-discriminating probe, the length is more restricted, as isdiscussed below.

In embodiments wherein the affinity pair comprises complementary nucleicacid regions that reversibly interact with one another, and cannothybridize or are not complementary to a target or template nucleic acidsequence, the complementary nucleic acid region sequences of theaffinity pair should be of sufficient length that under the conditionsof the assay and at the detection temperature, when the probe is notbound to a target, the structure of the probe is such that the bindingmoiety of the probe will not bind to the capture element, e.g., thecomplementary nucleic acid sequences are associated. Depending upon theassay conditions used, complementary nucleic acid sequences of 3-25nucleotide lengths can perform this function. An intermediate range of4-15, and more preferably 5-11, nucleotides is often appropriate. Theactual length will be chosen with reference to the target nucleic acidbinding sequence such that the secondary structure of the probe isstable when not bound to the target or template nucleic acid at thetemperature at which cleavage of a cleavage structure comprising theprobe bound to a target or template nucleic acid is performed. As thetarget nucleic acid binding sequence increases in size up to 100nucleotides, the length of the complementary nucleic acid sequences mayincrease up to 15-25 nucleotides. For a target or template nucleic acidbinding sequence greater than 100 nucleotides, the length of thecomplementary nucleic acid sequences are not increased further. If theprobe is also an allele-discriminating probe, the lengths of thecomplementary nucleic acid sequences are more restricted, as isdiscussed below.

According to this embodiment of the invention, allele-discriminatingprobes that do not hybridize sufficiently to a target-like nucleic acidsequence that contains one or more nucleotide mismatches as compared tothe target nucleic acid complementary sequence, must be designed suchthat, under the assay conditions used, reduction or elimination ofsecondary structure in the probe and hybridization with a target nucleicacid sequence will occur efficiently only when the target nucleic acidcomplementary sequence finds a perfectly complementary target sequenceunder certain reaction conditions (e.g., at a particular temperature orwithin a range of temperatures determined by experimental optimizationaccording to methods well known in the art to prevent efficient bindingof an allele-discriminating probe to a target-like or template-likenucleic acid sequence).

In one embodiment, an “allele-discriminating probe” according to theinvention refers to a probe that hybridizes to a target-like nucleicacid sequence that varies by at least one nucleotide from the targetnucleic acid sequence, wherein the variant nucleotide(s) is/are notlocated in the allele-discriminating site. According to this embodimentof the invention, “an allele-discriminating probe” cannot bindefficiently to a target-like nucleic acid sequence that also varies byat least one nucleotide in the allele-discriminating site (e.g., at aparticular temperature or within a range of temperatures determined byexperimental optimization according to methods well known in the art toprevent efficient binding of an allele-discriminating probe to atarget-like or template-like nucleic acid sequence).

In one embodiment of the invention, an allele discriminating probeaccording to the invention preferably comprises a target nucleic acidbinding sequence from 6 to 50 and preferably from 7 to 25 nucleotides,and complementary nucleic acid sequences from 3 to 8 nucleotides. Theguanosine-cytidine content of the secondary structure and probe-targethybrids, salt, and assay temperature should all be considered, forexample magnesium salts have a strong stabilizing effect that isparticularly important to consider when designing short,allele-discriminating probes.

If an allele-discriminating probe is to have a target nucleic acidbinding sequence near the upper limits of 50 nucleotides long, thesequence should be designed such that a single nucleotide mismatch to bediscriminated against occurs at or near the middle of the target nucleicacid complementary sequence. For example, probes comprising a sequencethat is 21 nucleotides long should preferably be designed so that themismatch occurs opposite one of the 14 most centrally locatednucleotides of the target nucleic acid complementary sequence and mostpreferably opposite one of the 7 most centrally located nucleotides.Designing a probe so that the mismatch to be discriminated againstoccurs in or near the middle of the target nucleic acid bindingsequence/target-like nucleic acid binding sequence is believed toimprove the performance of an allele-discriminating probe.

As used herein, “captured” as it refers to capture of a binding moietyby a capture element or capture of a tag by a capture element, meansspecifically bound by hydrogen bonding, covalent bonding, or via aninteraction between, for example a protein and a ligand, an antibody andan antigen, protein subunits, a nucleic acid binding protein and anucleic acid binding site, or between complementary nucleic acids,wherein one member of the interacting pair is attached to a solidsupport. Under conditions of stable capture, binding results in theformation of a heterodimer with a dissociation constant (K_(D)) of atleast about 1×10³ M⁻¹, usually at least 1×10⁴ M⁻¹, typically at least1×10⁵ M⁻¹, preferably at least 1×10⁶ M⁻¹ to 1×10⁷ M⁻¹ or more, undersuitable conditions.

Methods of performing binding reactions between a capture element, asdefined herein, and a binding moiety or tag, respectively, as definedherein, are well-known in the art and are described hereinbelow. Methodsof attaching a capture element according to the invention to a solidsupport, as defined herein, are well-known in the art and are definedhereinbelow.

Detection or verification of the labeled fragments or released flaps maybe accomplished by a variety of methods well known in the art and may bedependent on the characteristics of the labeled moiety or moietiescomprising a labeled cleavage structure. According to the method of theinvention wherein a probe comprises a secondary structure that changesupon binding to a target or template nucleic acid and further comprisesa binding moiety, the released labeled fragments or released flaps arecaptured by binding of a binding moiety to a capture element attached toa solid support.

a. Capture Element

A capture element, according to the invention can be any moiety thatspecifically binds (e.g. via hydrogen bonding or via an interactionbetween, for example a nucleic acid binding protein and a nucleic acidbinding site or between complementary nucleic acids) a binding moiety,as a result of attractive forces that exist between the binding moietyand the capture element. Methods of performing a reaction whereinspecific binding occurs between a capture element, as defined herein,and a binding moiety, as defined herein, are well-known in the art (seefor example, Sambrook et al., supra; Ausubel et al., supra). A captureelement, according to the invention can be any moiety that specificallybinds (e.g. via covalent or hydrogen bonding or electrostatic attractionor via an interaction between, for example a protein and a ligand, anantibody and an antigen, protein subunits, a nucleic acid bindingprotein and a nucleic acid binding site) a binding moiety, as a resultof attractive forces that exist between the tag and the capture element.Methods of performing a reaction wherein specific binding occurs betweena capture element, as defined herein, and a tag, as defined herein, arewell-known in the art, see for example, Sambrook et al., supra, Ausubelet al., supra. Specific binding only occurs when the secondary structureof the probe comprising a binding moiety has “changed”, as definedherein. Capture elements useful according to the invention include butare not limited to a nucleic acid binding protein or a nucleotidesequence. Capture elements useful according to the invention include butare not limited to biotin, streptavidin, a hapten, a protein, anucleotide sequence or a chemically reactive moiety.

In one embodiment of the invention, the reaction products, including thereleased labeled fragments, are subjected to size analysis. Methods fordetermining the size of a labeled fragment are known in the art andinclude, for example, gel electrophoresis, sedimentation in gradients,gel exclusion chromatography, mass spectroscopy, and homochromatography.

b. Solid Substrate

A solid substrate or solid support according to the invention is anysurface to which a molecule (e.g., a capture element) can beirreversibly bound, including but not limited to membranes, magneticbeads, tissue culture plates, silica based matrices, membrane basedmatrices, beads comprising surfaces including but not limited tostyrene, latex or silica based materials and other polymers for examplecellulose acetate, teflon, polyvinylidene difluoride, nylon,nitrocellulose, polyester, carbonate, polysulphone, metals, zeolites,paper, alumina, glass, polypropyle, polyvinyl chloride, polyvinylidenechloride, polytetrafluorethylene, polyethylene, polyamides, plastic,filter paper, dextran, germanium, silicon, (poly)tetrafluorethylene,gallium arsenide, gallium phosphide, silicon oxide, silicon nitrate andcombinations thereof. Useful solid substrates according to the inventionare also described in Sambrook et al., supra; Ausubel et al., supra;U.S. Pat. Nos. 5,427,779, 5,512,439, 5,589,586, 5,716,854 and 6,087,102;Southern et al., Nature Genetics Supplement, 21:5 and Joos et al., 1997,Analytical Biochemistry, 247:96.

Methods of attaching a capture element to a solid support are known inthe art and are described in Sambrook et al., supra, Ausubel et al.,supra, U.S. Pat. Nos. 5,427,779, 5,512,439, 5,589,586, 5,716,854 and6,087,102; Southern et al., supra and Joos et al., supra. Methods ofimmobilizing a nucleic acid sequence on a solid support are alsoprovided by the manufacturers of the solid support, e.g., for membranes:Pall Corporation, Schleicher & Schuell, for magnetic beads; Dyal, forculture plates: Costar, Nalgenenunc, and for other supports usefulaccording to the invention, CPG, Inc.

The amount of released labeled fragment, wherein “fragment” refers to areleased flap, as defined herein, and “released labeled fragment” refersto a released labeled flap, that is bound to a capture element attachedto a solid support can be measured while the labeled fragment remainsbound to the capture element or after release of the labeled fragmentfrom the capture element. Release of a labeled fragment from a captureelement is carried out by incubating labeled fragment-capture elementcomplexes in the presence of an excess amount of a competing, unlabeledfragment or by the addition of a buffer that inhibits binding of thelabeled fragment to the capture element, for example as a result of saltconcentration or pH of the buffer.

During or after amplification, separation of the released labeledfragments from, a reaction mixture can be accomplished by, for example,contacting the reaction mixture with a solid phase extractant (SPE). Forexample, materials having an ability to bind nucleic acids on the basisof size, charge, or interaction with the nucleic acid bases can be addedto the reaction mixture, under conditions where labeled, uncleavednucleic acids are bound and short, labeled fragments are not. Such SPEmaterials include ion exchange resins or beads, such as the commerciallyavailable binding particles Nensorb (DuPont Chemical Co.), Nucleogen(The Nest Group), PEI, BakerBond™ PEI, Amicon PAE 1,000, Selectacel™PEI, Boronate SPE with a 3′-ribose probe, SPE containing sequencescomplementary to the 3′-end of the probe, and hydroxylapatite. In aspecific embodiment, if a dual labeled oligonucleotide comprising abiotin label separated from a second label by a nuclease susceptiblecleavage site is employed as the signal means, the reaction mixture canbe contacted with materials containing a capture element such as avidinor streptavidin, or an antibody or monoclonal antibody to biotin, boundto a solid support, such as beads and particles, including magneticparticles.

Following the step in which a reaction mixture, for example a PCRmixture has been contacted with an SPE, the SPE material can be removedby filtration, sedimentation, or magnetic attraction, leaving thelabeled fragments free of uncleaved labeled oligonucleotides andavailable for detection.

c. Binding Moieties

A binding moiety according to the invention refers to a region of aprobe that is released upon cleavage of the probe by a nuclease andbinds specifically (via hydrogen binding with a complementary nucleicacid or via an interaction with a binding protein) to a capture elementas a result of attractive forces that exist between the binding moietyand the capture element, and wherein specific binding between thebinding moiety and the capture element only occurs when the secondarystructure of the probe has “changed”, as defined herein.

A “tag” refers to a moiety that is operatively linked to the 5′ end of aprobe (for example R in FIG. 7) and specifically binds to a captureelement as a result of attractive forces that exist between the tag andthe capture element, and wherein specific binding between the tag andthe capture element only occurs when the secondary structure of theprobe has changed (for example, such that the tag is accessible to acapture element). “Specifically binds” as it refers to a “tag” and acapture element means via covalent or hydrogen bonding or electrostaticattraction or via an interaction between, for example a protein and aligand, an antibody and an antigen, protein subunits, or a nucleic acidbinding protein and a nucleic acid binding site. Second binding moietiesinclude but are not limited to biotin, streptavidin, a hapten, aprotein, or a chemically reactive moiety.

Methods of incorporating a tag, as defined herein, into a nucleic acid(e.g., a probe according to the invention) are well known in the art andare described in Ausubel et al., supra, Sambrook et al., supra, and U.S.Pat. Nos. 5,716,854 and 6,087,102.

d. Target Nucleic Acid

The invention provides for a “target nucleic acid” that is apolynucleotide which comprises in 3′ to 5′ order a first region that isat least partially complementary to a first oligonucleotide, anextension region and a second region that is at least partiallycomplementary to a second oligonucleotide. The target nucleic acid maycomprise single or double-stranded DNA or RNA.

A target nucleic acid according to the invention comprises a “firstregion” that is a length of nucleotides sufficient to permithybridization and extension of a primer (e.g., a first oligonucleotideas defined herein) wherein the “first region” is at least partiallycomplementary to a primer (e.g., a first oligonucleotide as definedherein). A “first region” is in the range of about 6 nucleotides toabout 1000 nucleotides in length, with a preferred range of about 8-30nucleotides, and optimally, a range of 10-25 nucleotides.

A target nucleic acid according to the invention also comprises an“extension region” that is a length of nucleotides sufficient to permitextension of an oligonucleotide (e.g., a first or secondoligonucleotide) via a nucleic acid polymerization activity. An“extension region” is in the range of about 1 nucleotide to about 1000nucleotides in length, with a preferred range of about 3-100nucleotides, and optimally, a range of 3-30 nucleotides in length.

The second region of a target nucleic acid is a length of nucleotidesthat is at least partially complementary to a probe (e.g., a secondoligonucleotide, defined herein). A “second region” is in the range ofabout 6 nucleotides to about 1000 nucleotides in length, with apreferred range of about 8-30 nucleotides, and optimally, a range of10-25 nucleotides.

e. Template Nucleic Acid

The invention also provides for a “template nucleic acid” that is apolynucleotide which comprises in 3′ to 5′ order a first region that isat least partially complementary to a primer (e.g., the released flap ofa second oligonucleotide, defined herein), an extension region and asecond region that is at least partially complementary to a probe (e.g.,a third oligonucleotide, as defined herein).

The invention also provides for a “template nucleic acid” that is apolynucleotide which comprises in 3′ to 5′ order a first region that isat least partially complementary to a primer (e.g., the released flap ofa second oligonucleotide, defined herein below), a first extensionregion, a second region that is at least partially complementary to afirst probe (e.g., a third oligonucleotide, defined herein), a secondextension region, and a third region that is at least partiallycomplementary to a second probe (e.g., a third oligonucleotide, definedherein). The template nucleic acid may comprise single ordouble-stranded DNA or RNA.

A template nucleic acid according to the invention comprises a “firstregion” that is a length of nucleotides sufficient to permithybridization and extension of a primer (e.g., the released flap of asecond oligonucleotide as defined herein) wherein the “first region” isat least partially complementary to a primer (e.g., the released flap ofa second oligonucleotide as defined herein). A “first region” is in therange of about 6 nucleotides to about 1000 nucleotides in length, with apreferred range of about 8-30 nucleotides, and optimally, a range of10-25 nucleotides.

A template nucleic acid according to the invention also comprises an“extension region” that is a length of nucleotides sufficient to permitextension of an oligonucleotide (e.g., the released flap of a secondoligonucleotide as defined herein) via a nucleic acid polymerizationactivity. An “extension region” is in the range of about 1 nucleotide toabout 1000 nucleotides in length, with a preferred range of about 3-100nucleotides, and optimally, a range of 3-30 nucleotides in length.

The second region of a template nucleic acid is a length of nucleotidesthat is at least partially complementary to a probe (e.g., a thirdoligonucleotide, defined herein). A “second region” is in the range ofabout 6 nucleotides to about 1000 nucleotides in length, with apreferred range of about 8-30 nucleotides, and optimally, a range of10-25 nucleotides.

1. Labels

A template nucleic acid according to the invention can be labeled e.g.,by the attachment of a radiolabel, a fluorescent label a quencher or anyof the labels recited in the section IV, B. titled “How to Prepare aLabeled Cleavage Structure.” A labeled template nucleic acid is preparedaccording to methods well known in the art (see Sambrook et al., supra;Ausubel et al., supra) and described herein.

In one embodiment, the template nucleic acid is operable coupled to onemember of a pair of interactive labels. In a further embodiment, theinteractive label is a quencher or fluorescer. In yet another embodimentthe interactive, the label is operable coupled to the 5′ nucleotide ofthe template nucleic acid.

2. Solid Support

In one embodiment, a template nucleic acid is attached to a solidsupport, e.g., membrane, as taught in the Section entitled “SolidSubstrate”. The solid support may have multiple different templatenucleic acids attached in an array format. In this embodiment, thetemplate nucleic acid can be labeled with a first member of a pair ofinteractive labels, e.g., reporter or quencher group. A detectablesignal is generated by the cleavage and displacement of a thirdoligonucleotide which is operable coupled to a second member of a pairof interactive labels. In this embodiment, the reporter signal islocalized to the region of the solid support to which the specifictemplate nucleic acid is attached. Each different target nucleic acidcan produce a detectable signal indicative of the presence or amount ofa target nucleic acid.

f. Hybridization

Primers and probes according to the invention (e.g., first, second,third or fourth oligonucleotides or the released flap of a secondoligonucleotide) can be labeled and can be used to prepare a labeledcleavage structure. The following combinations of primers and probes canbe annealed to sequences within a target nucleic acid sequence: a secondoligonucleotide or a second oligonucleotide and first oligonucleotide.The following combinations of primers and probes can be annealed tosequences within a template nucleic acid: a third oligonucleotide, athird oligonucleotide and the released flap of a second oligonucleotide,a fourth oligonucleotide, a third and a fourth oligonucleotide, and athird oligonucleotide, a fourth oligonucleotide and the released flap ofa target nucleic acid.

In one embodiment, a hybridized first oligonucleotide is extended bypolymerization to form a cleavage structure and the cleavage structureis cleaved by a cleavage means, according to the invention, to release aflap of the second oligonucleotide. In another embodiment, a hybridizedreleased flap of a second oligonucleotide is extended by polymerizationto form a cleavage structure and the cleavage structure is cleaved by acleavage means, according to the invention, to release a flap of a thirdoligonucleotide, or flaps of a third and a fourth oligonucleotide, or torelease a third or a fourth oligonucleotide from a template nucleic acid

Typically, selective hybridization occurs when two nucleic acidsequences are substantially complementary (at least about 65%complementary over a stretch of at least 14 to 25 nucleotides,preferably at least about 75%, more preferably at least about 90%complementary). See Kanehisa, M., 1984, Nucleic Acids Res. 12: 203,incorporated herein by reference. As a result, it is expected that acertain degree of mismatch at the priming site of a primer, as definedherein, is tolerated. Such mismatch may be small, such as a mono-, di-or tri-nucleotide. Alternatively, a region of mismatch may encompassloops, which are defined as regions in which there exists a mismatch inan uninterrupted series of four or more nucleotides.

Numerous factors influence the efficiency and selectivity ofhybridization of an oligonucleotide probe to a second nucleic acidmolecule. These factors, which include oligonucleotide length,nucleotide sequence and/or composition, hybridization temperature,buffer composition and potential for steric hindrance in the region towhich the oligonucleotide is required to hybridize, will be consideredwhen designing oligonucleotide primers according to the invention.

A positive correlation exists between oligonucleotide length and boththe efficiency and accuracy with which an oligonucleotide will anneal toa target sequence. In particular, longer sequences have a higher meltingtemperature (T_(M)) than do shorter ones, and are less likely to berepeated within a given target sequence, thereby minimizing promiscuoushybridization. Oligonucleotide sequences with a high G-C content or thatcomprise palindromic sequences tend to self-hybridize, as do theirintended target sites, since unimolecular, rather than bimolecular,hybridization kinetics are generally favored in solution. However, it isalso important to design an oligonucleotide that contains sufficientnumbers of G-C nucleotide pairings since each G-C pair is bound by threehydrogen bonds, rather than the two that are found when A and T basespair to bind the target sequence, and therefore forms a tighter,stronger bond. Hybridization temperature varies inversely witholigonucleotide annealing efficiency, as does the concentration oforganic solvents, e.g., formamide, that might be included in a primingreaction or hybridization mixture, while increases in salt concentrationfacilitate binding. Under stringent annealing conditions, longerhybridization probes, or synthesis primers, hybridize more efficientlythan do shorter ones, which are sufficient under more permissiveconditions. As herein used, the term “standard stringent conditions”means hybridization will occur only if there is at least 95% andpreferably at least 97% identity between the sequences. The sequenceshybridize under stringent conditions following incubation of thesequences overnight at 42° C., followed by stringent washes (0.2×SSC at65° C.). Certain embodiments, for example wherein an allelediscriminating probe is used to discriminate against sequences thatvary, for example by at least one nucleotide, may require higherhybridization temperatures for specific hybridization. As severalfactors affect the stringency of hybridization, the combination ofparameters is more important than the absolute measure of a singlefactor.

g. Production of a Nucleic Acid

The invention provides nucleic acids to be detected and or measured, foramplification of a target nucleic acid sequence and for formation of acleavage structure.

The present invention utilizes nucleic acids comprising RNA, cDNA,genomic DNA, synthetic forms, and mixed polymers. The invention includesboth sense and antisense strands of a nucleic acid. According to theinvention, the nucleic acid may be chemically or biochemically modifiedor may contain non-natural or derivatized nucleotide bases. Suchmodifications include, for example, labels, methylation, substitution ofone or more of the naturally occurring nucleotides with an analog,internucleotide modifications such as uncharged linkages (e.g. methylphosphonates, phosphorodithioates, etc.), pendent moieties (e.g.,polypeptides), intercalators, (e.g. acridine, psoralen, etc.) chelators,alkylators, and modified linkages (e.g. alpha anomeric nucleic acids,etc.) Also included are synthetic molecules that mimic polynucleotidesin their ability to bind to a designated sequence via hydrogen bondingand other chemical interactions. Such molecules are known in the art andinclude, for example, those in which peptide linkages substitute forphosphate linkages in the backbone of the molecule.

1. Nucleic Acids Comprising DNA

a. Cloning

Nucleic acids comprising DNA can be isolated from cDNA or genomiclibraries by cloning methods well known to those skilled in the art(Ausubel et al., supra). Briefly, isolation of a DNA clone comprising aparticular nucleic acid sequence involves screening a recombinant DNA orcDNA library and identifying the clone containing the desired sequence.Cloning will involve the following steps. The clones of a particularlibrary are spread onto plates, transferred to an appropriate substratefor screening, denatured, and probed for the presence of a particularnucleic acid. A description of hybridization conditions, and methods forproducing labeled probes is included below.

The desired clone is preferably identified by hybridization to a nucleicacid probe or by expression of a protein that can be detected by anantibody. Alternatively, the desired clone is identified by polymerasechain amplification of a sequence defined by a particular set of primersaccording to the methods described below.

The selection of an appropriate library involves identifying tissues orcell lines that are an abundant source of the desired sequence.Furthermore, if a nucleic acid of interest contains regulatory sequenceor intronic sequence a genomic library is screened (Ausubel et al.,supra).

b. Genomic DNA

Nucleic acid sequences of the invention are amplified from genomic DNA.Genomic DNA is isolated from tissues or cells according to the followingmethod.

To facilitate detection of a variant form of a gene from a particulartissue, the tissue is isolated free from surrounding normal tissues. Toisolate genomic DNA from mammalian tissue, the tissue is minced andfrozen in liquid nitrogen. Frozen tissue is ground into a fine powderwith a prechilled mortar and pestle, and suspended in digestion buffer(100 mM NaCl, 10 mM Tris-HCl, pH 8.0, 25 mM EDTA, pH 8.0, 0.5% (w/v)SDS, 0.1 mg/ml proteinase K) at 1.2 ml digestion buffer per 100 mg oftissue. To isolate genomic DNA from mammalian tissue culture cells,cells are pelleted by centrifugation for 5 min at 500×g, resuspended in1-10 ml ice-cold PBS, repelleted for 5 min at 500×g and resuspended in 1volume of digestion buffer.

Samples in digestion buffer are incubated (with shaking) for 12-18 hoursat 50° C., and then extracted with an equal volume ofphenol/chloroform/isoamyl alcohol. If the phases are not resolvedfollowing a centrifugation step (10 min at 1700×g), another volume ofdigestion buffer (without proteinase K) is added and the centrifugationstep is repeated. If a thick white material is evident at the interfaceof the two phases, the organic extraction step is repeated. Followingextraction the upper, aqueous layer is transferred to a new tube towhich will be added ½ volume of 7.5M ammonium acetate and 2 volumes of100% ethanol. The nucleic acid is pelleted by centrifugation for 2 minat 1700×g, washed with 70% ethanol, air dried and resuspended in TEbuffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0) at 1 mg/ml. ResidualRNA is removed by incubating the sample for 1 hour at 37° C. in thepresence of 0.1% SDS and 1 μg/ml DNase-free RNase, and repeating theextraction and ethanol precipitation steps. The yield of genomic DNA,according to this method is expected to be approximately 2 mg DNA/1 gcells or tissue (Ausubel et al., supra). Genomic DNA isolated accordingto this method can be used for PCR analysis, according to the invention.

c. Restriction Digest (of cDNA or Genomic DNA)

Following the identification of a desired cDNA or genomic clonecontaining a particular target nucleic acid sequence, nucleic acids ofthe invention may be isolated from these clones by digestion withrestriction enzymes.

The technique of restriction enzyme digestion is well known to thoseskilled in the art (Ausubel et al., supra). Reagents useful forrestriction enzyme digestion are readily available from commercialvendors including Stratagene, as well as other sources.

d. PCR

Nucleic acids of the invention may be amplified from genomic DNA orother natural sources by the polymerase chain reaction (PCR). PCRmethods are well-known to those skilled in the art.

PCR provides a method for rapidly amplifying a particular DNA sequenceby using multiple cycles of DNA replication catalyzed by a thermostable,DNA-dependent DNA polymerase to amplify the target sequence of interest.PCR requires the presence of a target nucleic acid sequence to beamplified, two single stranded oligonucleotide primers flanking thesequence to be amplified, a DNA polymerase, deoxyribonucleosidetriphosphates, a buffer and salts.

PCR, is performed as described in Mullis and Faloona, 1987, MethodsEnzymol., 155: 335, herein incorporated by reference.

The polymerase chain reaction (PCR) technique, is disclosed in U.S. Pat.Nos. 4,683,202, 4,683,195 and 4,800,159. In its simplest form, PCR is anin vitro method for the enzymatic synthesis of specific DNA sequences,using two oligonucleotide primers that hybridize to opposite strands andflank the region of interest in the target DNA. A repetitive series ofreaction steps involving template denaturation, primer annealing and theextension of the annealed primers by DNA polymerase results in theexponential accumulation of a specific fragment whose termini aredefined by the 5′ ends of the primers. PCR is reported to be capable ofproducing a selective enrichment of a specific DNA sequence by a factorof 10⁹. The PCR method is also described in Saiki et al., 1985, Science230:1350.

PCR is performed using template DNA (at least 1 fg; more usefully,1-1000 ng) and at least 25 pmol of oligonucleotide primers. A typicalreaction mixture includes: 2 μl of DNA, 25 pmol of oligonucleotideprimer, 2.5 μl of a suitable buffer, 0.4 μl of 1.25 μM dNTP, 2.5 unitsof Taq DNA polymerase (Stratagene) and deionized water to a total volumeof 25 μl. Mineral oil is overlaid and the PCR is performed using aprogrammable thermal cycler.

The length and temperature of each step of a PCR cycle, as well as thenumber of cycles, are adjusted according to the stringency requirementsin effect. Annealing temperature and timing are determined both by theefficiency with which a primer is expected to anneal to a template andthe degree of mismatch that is to be tolerated. The ability to optimizethe stringency of primer annealing conditions is well within theknowledge of one of moderate skill in the art. An annealing temperatureof between 30° C. and 72° C. is used. Initial denaturation of thetemplate molecules normally occurs at between 92° C. and 99° C. for 4minutes, followed by 20-40 cycles consisting of denaturation (94-99° C.for 15 seconds to 1 minute), annealing (temperature determined asdiscussed above; 1-2 minutes), and extension (72° C. for 1 minute). Thefinal extension step is generally carried out for 4 minutes at 72° C.,and may be followed by an indefinite (0-24 hour) step at 4° C.

Detection methods generally employed in standard PCR techniques use alabeled probe with the amplified DNA in a hybridization assay.Preferably, the probe is labeled, e.g., with ³²P, biotin, horseradishperoxidase (HRP), etc., to allow for detection of hybridization.

Other means of detection include the use of fragment length polymorphism(PCR FLP), hybridization to allele-specific oligonucleotide (ASO) probes(Saiki et al., 1986, Nature 324:163), or direct sequencing via thedideoxy method (using amplified DNA rather than cloned DNA). Thestandard PCR technique operates (essentially) by replicating a DNAsequence positioned between two primers, providing as the major productof the reaction a DNA sequence of discrete length terminating with theprimer at the 5′ end of each strand. Thus, insertions and deletionsbetween the primers result in product sequences of different lengths,which can be detected by sizing the product in PCR-FLP. In an example ofASO hybridization, the amplified DNA is fixed to a nylon filter (by, forexample, UV irradiation) in a series of “dot blots”, then allowed tohybridize with an oligonucleotide probe labeled with HRP under stringentconditions. After washing, terramethylbenzidine (TMB) and hydrogenperoxide are added: HRP oxidizes the hydrogen peroxide, which in turnoxidizes the TMB to a blue precipitate, indicating a hybridized probe.

2. Nucleic Acids Comprising RNA

The present invention also provides a nucleic acid comprising RNA.

Nucleic acids comprising RNA can be purified according to methods wellknown in the art (Ausubel et al., supra). Total RNA can be isolated fromcells and tissues according to methods well known in the art (Ausubel etal., supra) and described below.

RNA is purified from mammalian tissue according to the following method.Following removal of the tissue of interest, pieces of tissue of ≦2 gare cut and quick frozen in liquid nitrogen, to prevent degradation ofRNA. Upon the addition of a suitable volume of guanidinium solution (forexample 20 ml guanidinium solution per 2 g of tissue), tissue samplesare ground in a tissuemizer with two or three 10-second bursts. Toprepare tissue guanidinium solution (1 L) 590.8 g guanidiniumisothiocyanate is dissolved in approximately 400 ml DEPC-treated H₂O. 25ml of 2 M Tris-HCl, pH 7.5 (0.05 M final) and 20 ml Na₂EDTA (0.01 Mfinal) is added, the solution is stirred overnight, the volume isadjusted to 950 ml, and 50 ml 2-ME is added.

Homogenized tissue samples are subjected to centrifugation for 10 min at12,000×g at 12° C. The resulting supernatant is incubated for 2 min at65° C. in the presence of 0.1 volume of 20% Sarkosyl, layered over 9 mlof a 5.7M CsCl solution (0.1 g CsCl/ml), and separated by centrifugationovernight at 113,000×g at 22° C. After careful removal of thesupernatant, the tube is inverted and drained. The bottom of the tube(containing the RNA pellet) is placed in a 50 ml plastic tube andincubated overnight (or longer) at 4° C. in the presence of 3 ml tissueresuspension buffer (5 mM EDTA, 0.5% (v/v) Sarkosyl, 5% (v/v) 2-ME) toallow complete resuspension of the RNA pellet. The resulting RNAsolution is extracted sequentially with 25:24:1phenol/chloroform/isoamyl alcohol, followed by 24:1 chloroform/isoamylalcohol, precipitated by the addition of 3 M sodium acetate, pH 5.2, and2.5 volumes of 100% ethanol, and resuspended in DEPC water (Chirgwin etal., 1979, Biochemistry, 18: 5294).

Alternatively, RNA is isolated from mammalian tissue according to thefollowing single step protocol. The tissue of interest is prepared byhomogenization in a glass teflon homogenizer in 1 ml denaturing solution(4M guanidinium thiosulfate, 25 mM sodium citrate, pH 7.0, 0.1M 2-ME,0.5% (w/v) N-laurylsarkosine) per 100 mg tissue. Following transfer ofthe homogenate to a 5-ml polypropylene tube, 0.1 ml of 2 M sodiumacetate, pH 4, 1 ml water-saturated phenol, and 0.2 ml of 49:1chloroform/isoamyl alcohol are added sequentially. The sample is mixedafter the addition of each component, and incubated for 15 min at 0-4°C. after all components have been added. The sample is separated bycentrifugation for 20 min at 10,000×g, 4° C., precipitated by theaddition of 1 ml of 100% isopropanol, incubated for 30 minutes at −20°C. and pelleted by centrifugation for 10 minutes at 10,000×g, 4° C. Theresulting RNA pellet is dissolved in 0.3 ml denaturing solution,transferred to a microfuge tube, precipitated by the addition of 0.3 mlof 100% isopropanol for 30 minutes at −20° C., and centrifuged for 10minutes at 10,000×g at 4° C. The RNA pellet is washed in 70% ethanol,dried, and resuspended in 100-200 μl DEPC-treated water or DEPC-treated0.5% SDS (Chomczynski and Sacchi, 1987, Anal. Biochem., 162: 156).

Nucleic acids comprising RNA can be produced according to the method ofin vitro transcription.

The technique of in vitro transcription is well known to those of skillin the art. Briefly, the gene of interest is inserted into a vectorcontaining an SP6, T3 or T7 promoter. The vector is linearized with anappropriate restriction enzyme that digests the vector at a single sitelocated downstream of the coding sequence. Following a phenol/chloroformextraction, the DNA is ethanol precipitated, washed in 70% ethanol,dried and resuspended in sterile water. The in vitro transcriptionreaction is performed by incubating the linearized DNA withtranscription buffer (200 mM Tris-HCl, pH 8.0, 40 mM MgCl₂, 10 mMspermidine, 250 NaCl [T7 or T3] or 200 mM Tris-HCl, pH 7.5, 30 mM MgCl₂,10 mM spermidine [SP6]), dithiothreitol, RNase inhibitors, each of thefour ribonucleoside triphosphates, and either SP6, T7 or T3 RNApolymerase for 30 min at 37° C. To prepare a radiolabeled polynucleotidecomprising RNA, unlabeled UTP will be omitted and ³⁵S-UTP will beincluded in the reaction mixture. The DNA template is then removed byincubation with DNaseI. Following ethanol precipitation, an aliquot ofthe radiolabeled RNA is counted in a scintillation counter to determinethe cpm/μl (Ausubel et al., supra).

Alternatively, nucleic acids comprising RNA are prepared by chemicalsynthesis techniques such as solid phase phosphoramidite (describedabove).

3. Nucleic Acids Comprising Oligonucleotides

A nucleic acid comprising oligonucleotides can be made by usingoligonucleotide synthesizing machines which are commercially available(described above).

It is well known by those with skill in the art that oligonucleotidescan be synthesized with certain chemical and/or capture moieties,(including capture elements as defined herein) such that they can becoupled to solid supports and bind to a binding moiety or a tag.Suitable capture elements include, but are not limited to a nucleic acidbinding protein or a nucleotide sequence, biotin, a hapten, a protein,or a chemically reactive moiety. Such oligonucleotides may either beused first in solution, and then captured onto a solid support, or firstattached to a solid support and then used in a detection reaction. Anexample of the latter would be to couple a upstream probe molecule to asolid support, such that the 5′ end of the upstream probe moleculecomprised a fluorescent quencher. The same upstream probe molecule wouldalso comprise a fluorophore in a location such that a FEN nucleasecleavage would physically separate the quencher from the fluorophore.For example, the target nucleic acid could hybridize with thesolid-phase upstream probe oligonucleotide, and a liquid phase upstreamprimer could also hybridize with the target molecule, such that a FENcleavage reaction occurs on the solid support and liberates the 5′quencher moiety from the complex. This would cause the solidsupport-bound fluorophore to be detectable, and thus reveal the presenceof a cleavage event upon a suitably labeled or identified solid support.Different upstream probe molecules could be bound to different locationson an array. The location on the array would identify the probemolecule, and indicate the presence of the template to which the probemolecule can hybridize.

II. Duplexes

The invention provides for duplexes that are used to prepare cleavagestructures according to the invention.

A duplex, according to the invention, is formed by mixing, in any order,the components of the duplex, under conditions that permit hybridizationof the components of the duplex and formation of the duplex. In oneembodiment, the duplex is formed by first hybridizing the target nucleicacid or the template nucleic acid with the probe or probes, and thenadding the primer. In another embodiment, the duplex is formed byhybridizing the target nucleic acid and/or template nucleic acidsimultaneously with one or more probes and primers. In one embodiment,the duplex is formed during an annealing step of a thermal cyclingreaction. Hybridization of a target or template nucleic acid with one ormore probes is performed under suitable conditions. Suitable conditionsinclude, for example, a temperature that permits denaturation of a probecomprising a secondary structure and the formation of hydrogen bondsbetween complementary bases of the target or template nucleic acid andthe probe or probes. In certain embodiments, a suitable amount of adenaturing agent, such as dimethylsulfoxide (DMSO) or glycerol is addedto the hybridization mixture. A suitable amount of a denaturing agent issufficient to permit hybridization and formation of a first or secondduplex, according to the invention, as well as subsequent steps ofpolymerization and cleavage, described below. A concentration of adenaturing agent that is useful according to the invention will varydepending on the base pair compositions of the components of the duplex.A concentration of a denaturing agent that is useful according to theinvention will be determined experimentally by methods known in the artand described herein, to be sufficient to permit hybridization ofcomplementary nucleic acids, polymerization of a primer (e.g., a firstoligonucleotide or the released flap of a second oligonucleotide) andcleavage of a cleavage structure to release flaps, according to theinvention. In one embodiment, the denaturing agent is DMSO, used atconcentration of 0 to 6%, and preferably around 1.5 to 2% for nucleicacids in the range of approximately 0.1 to 1 kb. A concentration of DMSOgreater than 2% may be used for nucleic acids greater than 10 kb.Alternatively, glycerol can be used as a denaturing agent at aconcentration of from 0 to 10%, and preferably 5 to 8%. Both, or evenother denaturing agents, may be used in combination at concentrationsthat are determined experimentally by methods known in the art.

Methods of determining “conditions that permit hybridization” ofcomponents of a duplex are known in the art, and parameters thatinfluence nucleic acid hybridization are discussed in detail in thesection entitled “Nucleic Acids”.

A. First Duplex

The invention provides for a first duplex comprising a target nucleicacid (e.g., A′B′C′, FIG. 2; A′B′ C′, FIG. 17A), a primer (e.g., a firstoligonucleotide, e.g., A, FIG. 2 or FIG. 17A) and a probe (e.g., asecond oligonucleotide, e.g., FC, FIG. 2 or FIG. 17A). In oneembodiment, the probe (e.g., a second oligonucleotide) comprises a 5′region that is not complementary to the target nucleic acid. Accordingto this embodiment, a duplex is formed such that the 5′ region of theprobe is a flap and the extension region of the target nucleic acid issingle-stranded. In a second embodiment, the primer and the 5′ region ofthe probe hybridize to non-overlapping regions of the target nucleicacid, as defined herein. According to this embodiment, a duplex isformed such that the 5′ region of the second oligonucleotide is not aflap, and the extension region of the target nucleic acid is completelysingle-stranded, is partially single stranded (that is, less than 100%(e.g., 99%, 90%, 75%, 50%, 25%, 5%, etc. . . .) of the extension regionis single-stranded), or is completely single stranded. A duplexcomprising an extension region that is partially single stranded orcompletely double-stranded further comprises the following: 1. a primerand probe wherein one or both of the primer and probe is/are partiallycomplementary, defined hereinabove, to the extension region; or 2. aprimer and a probe wherein one or both of the primer and probe is/arecompletely complementary to the extension region. Formation of acleavage structure from a duplex wherein the 5′ region of the probe ishybridized to the target nucleic acid requires a nucleic acidpolymerization activity that possesses strand displacement activity.

In one embodiment, a duplex is formed by first hybridizing the targetnucleic acid with the probe and then adding the primer.

In another embodiment, a duplex is formed by simultaneously hybridizingthe target nucleic acid with the probe and primer.

B. Second Duplex

The invention provides for a second duplex comprising a template nucleicacid (e.g., F′G′H′, FIG. 2 or FIG. 17D), a primer (e.g., the releasedflap of a second oligonucleotide, e.g., F, FIG. 2 or FIG. 17D) and aprobe (e.g., a third oligonucleotide, e.g., FH, FIG. 2; H, FIG. 17D). Inone embodiment, the third oligonucleotide comprises a 5′ region that isnot complementary to the template nucleic acid. According to thisembodiment, a duplex is formed such that the 5′ region of the thirdoligonucleotide is a flap and the extension region of the templatenucleic acid is single-stranded. In a second embodiment, the primer(e.g., the released flap of a second oligonucleotide) and a probe (e.g.,a third oligonucleotide) hybridize to non-overlapping regions of thetemplate nucleic acid, defined herein. According to this embodiment, aduplex if formed such that the 5′ region of the probe (e.g., the thirdoligonucleotide) is not a flap, and the extension region of the targetnucleic acid is completely single stranded, is partially single stranded(that is, less than 100% (e.g., 99%, 90%, 75%, 50%, 25%, 5%, etc . . . )of the extension region is single-stranded), or is completely singlestranded. A duplex comprising an extension region that is partiallysingle stranded or completely double-stranded further comprises thefollowing: 1. a primer and a probe wherein one or both of the primer andprobe is/are partially complementary, defined hereinabove, to theextension region; or 2. a primer and a probe wherein one or both of theprimer and probe is/are completely complementary to the extensionregion.

The invention also provides for a second duplex comprising a templatenucleic acid (e.g., F′G1′H1′G2′H2′, FIG. 5), a primer (e.g., thereleased flap of a second oligonucleotide, e.g., F, FIG. 5) a firstprobe (e.g., a third oligonucleotide, e.g., FH1, FIG. 5) and a fourthprobe (e.g., a fourth oligonucleotide, e.g., FH2, FIG. 5). In oneembodiment, the first probe comprises a 5′ region that is notcomplementary to the template nucleic acid. In another embodiment, thesecond probe comprises a 5′ region that is not complementary to thetemplate nucleic acid. In another embodiment, both the first and secondprobes comprise a 5′ region that is not complementary to the templatenucleic acid. According to these embodiments, a duplex is formed suchthat the 5′ region of the first probe, the second probe, or both thefirst and second probe, is/are a flap(s) and the extension regions ofthe template nucleic acid are single-stranded. In a second embodiment,the primer (e.g., the released flap of a second oligonucleotide) and the5′ region of the first probe (e.g., a third oligonucleotide), or the 5′region of the second probe (e.g., a fourth oligonucleotide) or the 5′regions of both the first and second probe hybridize to non-overlappingregions of the template nucleic acid. According to this embodiment, aduplex is formed such that the 5′ region of the first probe (e.g., thethird oligonucleotide) or the 5′ region of the second probe, or the 5′regions of both the first and second probe is/are not a flap, and theextension regions of the target nucleic acid are single stranded, arepartially single stranded (that is, less than 100% (e.g., 99%, 90%, 75%,50%, 25%, 5%, etc. . . .) of the extension regions are single-stranded),or are completely single stranded. A duplex comprising an extensionregion that is partially single stranded or completely double-strandedfurther comprises either a primer and two probes wherein at least one ofthe primer and probes is/are partially complementary, definedhereinabove, to the extension region(s), or a primer and two probeswherein at least one of the primer and probes is/are completelycomplementary to the extension region(s).

Formation of a cleavage structure from a duplex wherein the 5′ region ofthe first and/or second probe is hybridized to the template nucleic acidrequires a nucleic acid polymerization activity that possesses stranddisplacement activity. In one embodiment, a second duplex is formed byfirst hybridizing the template nucleic acid with the first probe or withboth the first and second probes, prior to the addition of the primer.In an another embodiment, a second duplex is formed by hybridizing thetemplate nucleic acid with both the probes and primers simultaneously.

III. Nucleic Acid Polymerization Activities

The invention provides for nucleic acid polymerization activities(including nucleic acid polymerases) that are useful in an isothermalreaction. A nucleic acid polymerization activity that is useful in anisothermal reaction according to the invention includes, but is notlimited to any of the nucleic acid polymerases listed below.

A nucleic acid polymerase according to the invention can bethermostable. As used herein, “thermostable” refers to an enzyme whichis stable and active at temperatures as great as preferably betweenabout 90-100° C. and more preferably between about 70-98° C. to heat ascompared, for example, to a non-thermostable form of an enzyme with asimilar activity. For example, a thermostable nucleic acid polymerase orFEN nuclease derived from thermophilic organisms such as P. furiosus, M.jannaschii, A. fulgidus or P. horikoshii are more stable and active atelevated temperatures as compared to a nucleic acid polymerase from E.coli or a mammalian FEN enzyme. A representative thermostable nucleicacid polymerase isolated from Thermus aquaticus (Taq) is described inU.S. Pat. No. 4,889,818 and a method for using it in conventional PCR isdescribed in Saiki et al., 1988, Science 239:487. Another representativethermostable nucleic acid polymerase isolated from P. furiosus (Pfu) isdescribed in Lundberg et al., 1991, Gene, 108:1-6. Additionalrepresentative temperature stable polymerases include, e.g., polymerasesextracted from the thermophilic bacteria Thermus flavus, Thermus ruber,Thermus thermophilus, Bacillus stearothermophilus (which has a somewhatlower temperature optimum than the others listed), Thermus lacteus,Thermus rubens, Thermotoga maritima, or from thermophilic archaeaThermococcus litoralis, and Methanothermus fervidus.

Temperature stable polymerases and FEN nucleases are preferred in athermocycling process wherein double stranded nucleic acids aredenatured by exposure to a high temperature (about 950 C) during the PCRcycle.

Known DNA polymerases useful according to the invention include, forexample, E. coli DNA polymerase I, Thermus thermophilus (Tth) DNApolymerase, Bacillus stearothermophilus DNA polymerase, Thermococcuslitoralis DNA polymerase, Thermus aquaticus (Taq) DNA polymerase andPyrococcus furiosus (Pfu) DNA polymerase.

Nucleic acid polymerases substantially lacking 5′ to 3′ exonucleaseactivity useful according to the invention include but are not limitedto Klenow and Klenow exo-, and T7 DNA polymerase (Sequenase).

Thermostable nucleic acid polymerases substantially lacking 5′ to 3′exonuclease activity useful according to the invention include but arenot limited to Pfu, exo- Pfu (a mutant form of Pfu that lacks 3′ to 5′exonuclease activity), the Stoffel fragment of Taq, N-truncated Bst,N-truncated Bca, Genta, JdF3 exo-, Vent, Vent exo- (a mutant form ofVent that lacks 3′ to 5′ exonuclease activity), Deep Vent, Deep Ventexo- (a mutant form of Deep Vent that lacks 3′ to 5′ exonucleaseactivity), U1Tma, and ThermoSequenase.

Nucleic acid polymerases useful in certain embodiments of the inventionsubstantially lack 3′ to 5′ exonuclease activity and include but are notlimited to exo- Pfu DNA polymerase (a mutant form of Pfu DNA polymerasethat substantially lacks 3′ to 5′ exonuclease activity, Cline et al.,1996, Nucleic Acids Research, 24: 3546; U.S. Pat. No. 5,556,772;commercially available from Stratagene, La Jolla, Calif. Catalogue #600163), exo- Tma DNA polymerase (a mutant form of Tma DNA polymerasethat substantially lacks 3′ to 5′ exonuclease activity), exo-Tli DNApolymerase (a mutant form of Tli DNA polymerase that substantially lacks3′ to 5′ exonuclease activity New England Biolabs, (Cat #257)), exo- E.coli DNA polymerase (a mutant form of E. coli DNA polymerase thatsubstantially lacks 3′ to 5′ exonuclease activity) exo- Klenow fragmentof E. coli DNA polymerase I (Stratagene, Cat #600069), exo- T7 DNApolymerase (a mutant form of T7 DNA polymerase that substantially lacks3′ to 5′ exonuclease activity), exo- KOD DNA polymerase (a mutant formof KOD DNA polymerase that substantially lacks 3′ to 5′ exonucleaseactivity), exo- JDF-3 DNA polymerase (a mutant form of JDF-3 DNApolymerase that substantially lacks 3′ to 5′ exonuclease activity),exo-PGB-D DNA polymerase (a mutant form of PGB-D DNA polymerase thatsubstantially lacks 3′ to 5′ exonuclease activity) New England Biolabs,Cat. #259, Tth DNA polymerase, Taq DNA polymerase (e.g., Cat. Nos.600131, 600132, 600139, Stratagene); UlTma (N-truncated) Thermatogamartima DNA polymerase; Klenow fragment of DNA polymerase I, 9° Nm DNApolymerase (discontinued product from New England Biolabs, Beverly,Mass.), “3′-5′ exo reduced” mutant (Southworth et al., 1996, Proc. Natl.Acad. Sci 93:5281) and Sequenase (USB, Cleveland, Ohio). The polymeraseactivity of any of the above enzyme can be defined by means well knownin the art. One unit of DNA polymerase activity, according to thesubject invention, is defined as the amount of enzyme which catalyzesthe incorporation of 10 nmoles of total dNTPs into polymeric form in 30minutes at optimal temperature.

Nucleic acid polymerases useful according to the invention include bothnative polymerases as well as polymerase mutants, which lack 5′ to 3′exonuclease activity. Nucleic acid polymerases useful according to theinvention can possess different degrees of thermostability. Preferably,a nucleic acid polymerase according to the invention exhibits stranddisplacement activity at the temperature at which it can extend anucleic acid primer. In a preferred embodiment of the invention, anucleic acid polymerase lacks both 5′ to 3′ and 3′ to 5′ exonucleaseactivity.

Additional nucleic acid polymerases substantially lacking 5′ to 3′exonuclease activity with different degrees of thermostability usefulaccording to the invention are listed below.

A. Bacteriophage DNA Polymerases (Useful for 37° C. Assays):

Bacteriophage DNA polymerases are devoid of 5′ to 3′ exonucleaseactivity, as this activity is encoded by a separate polypeptide.Examples of suitable DNA polymerases are T4, T7, and φ29 DNA polymerase.The enzymes available commercially are: T4 (available from many sourcese.g., Epicentre) and T7 (available from many sources, e.g. Epicentre forunmodified and USB for 3′ to 5′ exo⁻ T7 “Sequenase” DNA polymerase).

B. Archaeal DNA Polymerases:

There are 2 different classes of DNA polymerases which have beenidentified in archaea: 1. Family B/pol α type (homologs of Pfu fromPyrococcus furiosus) and 2. pol II type (homologs of P. furiosus DP1/DP22-subunit polymerase). DNA polymerases from both classes have been shownto naturally lack an associated 5′ to 3′ exonuclease activity and topossess 3′ to 5′ exonuclease (proofreading) activity. Suitable DNApolymerases (pol α or pol II) can be derived from archaea with optimalgrowth temperatures that are similar to the desired assay temperatures.Examples of suitable archaea include, but are not limited to:

1. Thermolabile (useful for 37° C. assays)—e.g., Methanococcus voltae

2. Thermostable (useful for non-PCR assays)—e.g., Sulfolobussolfataricus, Sulfolobus acidocaldarium, Methanococcus jannaschi,Thermoplasma acidophilum. It is estimated that suitable archaea exhibitmaximal growth temperatures of ≦80-85° C. or optimal growth temperaturesof ≦70-80° C.

3. Thermostable (useful for PCR assays)—e.g., Pyrococcus species(furiosus, species GB-D, species strain KOD1, woesii, abysii,horikoshii), Thermococcus species (litoralis, species 9° North-7,species JDF-3, gorgonarius), Pyrodictium occultum, and Archaeoglobusfulgidus. It is estimated that suitable archaea would exhibit maximalgrowth temperatures of ≧80-85° C. or optimal growth temperatures of≧70-80° C. Appropriate PCR enzymes from the archaeal pol α DNApolymerase group are commercially available, including KOD (Toyobo), Pfx(Life Technologies, Inc.), Vent (New England BioLabs), Deep Vent (NewEngland BioLabs), and Pwo (Boehringer-Mannheim).

Additional archaea related to those listed above are described in thefollowing references: Archaea: A Laboratory Manual (Robb, F. T. andPlace, A. R., eds.), Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1995 and Thermophilic Bacteria (Kristjansson, J. K., ed.)CRC Press, Inc., Boca Raton, Fla., 1992.

C. Eubacterial DNA Polymerases:

There are 3 classes of eubacterial DNA polymerases, pol I, II, and III.Enzymes in the Pol I DNA polymerase family possess 5′ to 3′ exonucleaseactivity, and certain members also exhibit 3′ to 5′ exonucleaseactivity. Pol II DNA polymerases naturally lack 5′ to 3′ exonucleaseactivity, but do exhibit 3′ to 5′ exonuclease activity. Pol III DNApolymerases represent the major replicative DNA polymerase of the celland are composed of multiple subunits. The pol III catalytic subunitlacks 5′ to 3′ exonuclease activity, but in some cases 3′ to 5′exonuclease activity is located in the same polypeptide.

There are no commercial sources of eubacterial pol II and pol III DNApolymerases.

There are a variety of commercially available Pol I DNA polymerases,some of which have been modified to reduce or abolish 5′ to 3′exonuclease activity. Methods used to eliminate 5′ to 3′ exonucleaseactivity of pol I DNA polymerases include:

-   -   mutagenesis (as described in Xu et al., 1997, J. Mol. Biol.,        268:284 and Kim et al., 1997, Mol. Cells, 7:468).    -   N-truncation by proteolytic digestion (as described in Klenow et        al., 1971, Eur. J. Biochem., 22: 371), or    -   N-truncation by cloning and expressing as C-terminal fragments        (as described in Lawyer et al., 1993, PCR Methods Appl., 2:275).

As for archaeal sources, the assay-temperature requirements determinewhich eubacteria should be used as a source of a DNA polymerase usefulaccording to the invention (e.g., mesophiles, thermophiles,hyperthermophiles).

1. Mesophilic/Thermolabile (Useful for 37° C. Assays)

-   -   i. DNA polymerases naturally substantially lacking 5′ to 3′        exonuclease activity: pol II or the pol III catalytic subunit        from mesophilic eubacteria, such as Escherchia coli,        Streptococcus pneumoniae, Haemophilus influenza, Mycobacterium        species (tuberculosis, leprae    -   ii. DNA polymerase mutants substantially lacking 5′ to 3′        exonuclease activity: Pol I DNA polymerases for N-truncation or        mutagenesis can be isolated from the mesophilic eubacteria        listed above (Ci). A commercially-available eubacterial DNA        polymerase pol I fragment is the Klenow fragment (N-truncated E.        coli pol I; Stratagene).

2. Thermostable (Useful for Non PCR Assays)

-   -   i. DNA polymerases naturally substantially lacking 5′ to 3′        exonuclease activity: Pol II or the pol III catalytic subunit        from thermophilic eubacteria, such as Bacillus species (e.g.,        stearothermophilus, caldotenax, caldovelox)    -   ii. DNA polymerase mutants substantially lacking 5′ to 3′        exonuclease activity: Suitable pol I DNA polymerases for        N-truncation or mutagenesis can be isolated from thermophilic        eubacteria such as the Bacillus species listed above.        Thermostable N-truncated fragments of B. stearothermophilus DNA        polymerase pol I are commercially available and sold under the        trade names Bst DNA polymerase I large fragment (Bio-Rad and        Isotherm DNA polymerase (Epicentre)). A C-terminal fragment of        Bacillus caldotenax pol I is available from Panvera (sold under        the tradename Ladderman).

3. Thermostable (Useful for PCR Assays)

-   -   i. DNA polymerases naturally substantially lacking 5′ to 3′        exonuclease activity: Pol II or pol III catalytic subunit from        Thermus species (aquaticus, thermophilus, flavus, ruber,        caldophilus, filiformis, brokianus) or from Thermotoga maritima.        The catalytic pol III subunits from Thermus thermophilus and        Thermus aquaticus are described in Yi-Ping et al., 1999, J. Mol.        Evol., 48:756 and McHenry et al., 1997, J. Mol. Biol., 272:178.    -   ii. DNA polymerase mutants substantially lacking 5′ to 3′        exonuclease activity: Suitable pol I DNA polymerases for        N-truncation or mutagenesis can be isolated from a variety of        thermophilic eubacteria, including Thermus species and        Thermotoga maritima (see above). Thermostable fragments of        Thermus aquaticus DNA polymerase pol I (Taq) are commercially        available and sold under the trade names KlenTaq1 (Ab Peptides),        Stoffel fragment (Perkin-Elmer), and ThermoSequenase (Amersham).        In addition to C-terminal fragments, 5′ to 3′ exonuclease⁻ Taq        mutants are also commercially available, such as TaqFS        (Hoffman-LaRoche). In addition to 5′-3′ exonuclease⁻ versions of        Taq, an N-truncated version of Thermotoga maritima DNA        polymerase I is also commercially available (tradename UlTma,        Perkin-Elmer).

Additional eubacteria related to those listed above are described inThermophilic Bacteria (Kristjansson, J. K., ed.) CRC Press, Inc., BocaRaton, Fla., 1992.

D. Eukaryotic 5′ to 3′ Exonuclease⁻ DNA Polymerases (Useful for 37° C.Assays)

There are several DNA polymerases that have been identified ineukaryotes, including DNA pol α (replication/repair), δ (replication), ε(replication), β (repair) and γ (mitochondrial replication). EukaryoticDNA polymerases are devoid of 5′ to 3′ exonuclease activity, as thisactivity is encoded by a separate polypeptide (e.g., mammalian FEN-1 oryeast RAD2). Suitable thermolabile DNA polymerases may be isolated froma variety of eukaryotes (including but not limited to yeast, mammaliancells, insect cells, Drosophila) and eukaryotic viruses (e.g., EBV,adenovirus).

It is possible that DNA polymerase mutants lacking 3′-5′ exonuclease(proofreading) activity, in addition to lacking 5′ to 3′ exonucleaseactivity, could exhibit improved performance in FEN-based detectionstrategies. For example, reducing or abolishing inherent 3′ to 5′exonuclease activity may lower background signals by diminishingnon-specific exonucleolytic degradation of labeled probes. Three 3′ to5′ exonuclease motifs have been identified, and mutations in theseregions have been shown to abolish 3′ to 5′ exonuclease activity inKlenow, φ29, T4, T7, and Vent DNA polymerases, yeast Pol α, Pol β, andPol γ, and Bacillus subtilis Pol III (reviewed in Derbeyshire et al.,1995, Methods. Enzymol. 262:363). Methods for preparing additional DNApolymerase mutants, with reduced or abolished 3′ to 5′ exonucleaseactivity, are well known in the art.

Commercially-available enzymes that lack both 5′ to 3′ and 3′ to 5′exonuclease activities include Sequenase (exo⁻ T7; USB), Pfu exo⁻(Stratagene), exo⁻ Vent (New England BioLabs), exo⁻ DeepVent (NewEngland BioLabs), exo⁻ Klenow fragment (Stratagene), Bst (Bio-Rad),Isotherm (Epicentre), Ladderman (Panvera), KlenTaq1 (Ab Peptides),Stoffel fragment (Perkin-Elmer), ThermoSequenase (USB), and TaqFS(Hoffman-LaRoche).

Nucleic acid polymerases with strand displacement activity are alsouseful according to the invention.

If polymerases other than Pfu are used, buffers and extensiontemperatures are selected to allow for optimal activity by theparticular polymerase useful according to the invention. Buffers andextension temperatures useful for polymerases according to the inventionare know in the art and can also be determined from the Vendor'sspecifications.

Additional nucleases useful according to the invention include a mutantform of Taq polymerase that lacks a 5′ to 3′ exonuclease activity butthat possesses a 3′ to 5′ DNA synthetic activity comprises the followingmutation: D144S/F667Y Taq wherein D144S eliminates 5′ to 3′ exonucleaseactivity and F667Y improves ddNTP incorporation.

Exo- mutants of PolI polymerase can be prepared according to the methodof Xu et al., 1997, J. Mol. Biol., 268: 284.

IV. Cleavage Structure

The invention provides for a cleavage structure that can be cleaved by anuclease (e.g., a FEN nuclease) and therefore teaches methods ofpreparing a cleavage structure.

A. Preparation of a Cleavage Structure

1. In one embodiment of the invention, a first cleavage structure isformed by incubating a target nucleic acid (A′B′C′, FIG. 2 or FIG. 17A),a downstream probe (e.g., a second oligonucleotide, e.g., FC, FIG. 2 orFIG. 17A) comprising a 5′ region that is not complementary to the targetnucleic acid, and an upstream primer (e.g., a first oligonucleotide,e.g., A, FIG. 2 or FIG. 17A) located not more than 1000 nucleotides fromthe probe, with a suitable buffer (for example Sentinel Molecular BeaconPCR core buffer (Catalog #600500), 10×Pfu buffer available fromStratagene (Catalog #200536), or 1× FullVelocity QPCR master mixavailable from Stratagene (Catalog#600561), under conditions that allowthe target nucleic acid sequence to hybridize to the oligonucleotides(for example 95° C. for 10 sec-5 minutes followed by cooling to betweenapproximately 50-60° C.) to form a duplex according to the invention.The 5′ region of the probe is a flap and the extension region of thetarget nucleic acid is single-stranded. The optimal temperature willvary depending on the specific probe(s), primers and polymerases. In oneembodiment, the 3′ end of the upstream primer is extended by a nucleicacid polymerization activity according to the invention such that thenewly synthesized 3′ end of the upstream oligonucleotide primer isadjacent to, as defined herein, the flap of the downstream probe.Extension can be carried out in the presence of 1× Sentinel Molecularcore buffer, 1×Pfu buffer, or 1× FullVelocity QPCR master mix for 15seconds at 72° C. The extension reaction can also be carried out for 30seconds at 60° C. utilizing the FullVelocity Q-PCR kit.

2. In another embodiment of the invention, a first cleavage structure isformed by incubating a target nucleic acid (A′B′C′, FIG. 2 or FIG. 17A),a downstream probe (e.g., a second oligonucleotide, e.g., FC, FIG. 2 orFIG. 17A) comprising a 5′ region that is complementary to the targetnucleic acid, and a upstream primer (e.g., a first oligonucleotide,e.g., A, FIG. 2; FIG. 17A) located not more than 1000 nucleotides fromthe probe, with a suitable buffer (for example Sentinel Molecular BeaconPCR core buffer (Catalog #600500), 10×Pfu buffer available fromStratagene (Catalog #200536) or 1× FullVelocity QPCR master mixavailable from Stratagene (Catalog#600561), under conditions that allowthe target nucleic acid sequence to hybridize to the oligonucleotides(for example 95° C. for 10 sec.-5 minutes followed by cooling to betweenapproximately 50-60° C.) to form a duplex according to the invention,wherein the 5′ region of the probe is hybridized to the target nucleicacid and the extension region of the target nucleic acid issingle-stranded. The optimal temperature will vary depending on thespecific probe(s), primers and polymerases. The 3′ end of the upstreamprimer is extended by a nucleic acid polymerization activity with stranddisplacement activity, according to the invention, such that the newlysynthesized 3′ end of the upstream oligonucleotide primer partiallydisplaces the 5′ end of the downstream oligonucleotide probe. Extensioncan be carried out in the presence of 1× Sentinel Molecular core buffer,1×Pfu buffer or 1× FullVelocity QPCR master mix for 15 seconds at 72° C.The extension reaction can also be carried out for 30 seconds at 60° C.utilizing the FullVelocity Q-PCR kit.

In one embodiment, a duplex is formed by first hybridizing the targetnucleic acid with the probe, and then adding the primer.

3. In one embodiment of the invention, a second cleavage structure isformed by incubating a template nucleic acid (F′G′H′, FIG. 2 or FIG.17D), a downstream probe (e.g., a third oligonucleotide, e.g., FH, FIG.2 or FIG. 17D) comprising a 5′ region that is not complementary to thetemplate nucleic acid, and an upstream primer (e.g., the released flapof a second oligonucleotide, e.g., F, FIG. 2 or FIG. 17D) located notmore than 1000 nucleotides from the probe, with a suitable buffer (forexample Sentinel Molecular Beacon PCR core buffer (Catalog #600500),10×Pfu buffer available from Stratagene (Catalog #200536), or 1×FullVelocity QPCR master mix available from Stratagene (Catalog#600561)under conditions that allow the template nucleic acid sequence tohybridize to the oligonucleotides (for example 95° C. for 10 sec.-5minutes followed by cooling to between approximately 50-60° C.) to forma second duplex according to the invention, wherein the 5′ region of theprobe is a flap and the extension region of the target nucleic acid isnot hybridized to the flap. The optimal temperature will vary dependingon the specific probe(s), primers and polymerases. In one embodiment,the 3′ end of the upstream primer is extended by a nucleic acidpolymerization activity according to the invention such that the newlysynthesized 3′ end of the upstream oligonucleotide primer is adjacentto, as defined herein, the flap of the downstream probe. Extension ispreferably carried out in the presence of 1× Sentinel Molecular corebuffer, 1×Pfu buffer or 1× FullVelocity QPCR master mix for 15 secondsat 72° C. The extension reaction can be carried out for 30 seconds at60° C. utilizing the FullVelocity Q-PCR kit.

4. In another embodiment of the invention, a second cleavage structureis formed by incubating a target nucleic acid (F′G′H′, FIG. 2; FIG.17D), a downstream probe (e.g., a third oligonucleotide, e.g., FH, FIG.2 or H, FIG. 17D) comprising a 5′ region that is complementary to thetarget nucleic acid, and a upstream primer (e.g., the released flap of asecond oligonucleotide, e.g., F, FIG. 2 or FIG. 17D) located not morethan 1000 nucleotides from the probe, with a suitable buffer (forexample Sentinel Molecular Beacon PCR core buffer (Catalog #600500),10×Pfu buffer available from Stratagene (Catalog #200536), or 1×FullVelocity QPCR master mix available from Stratagene (Catalog#600561),under conditions that allow the target nucleic acid sequence tohybridize to the oligonucleotides (for example 95° C. for 10 sec-5minutes followed by cooling to between approximately 50-60° C.) to forma duplex according to the invention, wherein the 5′ region of the probeis hybridized to the target nucleic acid and the extension region of thetarget nucleic acid is single-stranded. The optimal temperature willvary depending on the specific probe(s), primers and polymerases. The 3′end of the upstream primer is extended by a nucleic acid polymerizationactivity with strand displacement activity, according to the invention,such that the newly synthesized 3′ end of the upstream oligonucleotideprimer partially displaces the downstream oligonucleotide probe.Extension is preferably carried out in the presence of 1× SentinelMolecular core buffer, 1×Pfu buffer or 1× FullVelocity QPCR master mixavailable from Stratagene Calif. (Catalog#600561) for 15 seconds at 72°C. The extension reaction can also be carried out for 30 seconds at 60°C. utilizing the FullVelocity Q-PCR kit.

In one embodiment, a duplex is formed by first hybridizing the templatenucleic acid with the probe, and then adding the primer.

In another embodiment, a duplex is formed by hybridizing the templatenucleic acid with the probe and primer simultaneously.

5. In another embodiment of the invention, a second cleavage structureis formed by incubating a template nucleic acid (e.g., F′G1′H1′G2′H2′,FIG. 5) comprising first and second extension regions, a downstreamfirst probe (e.g., a third oligonucleotide, e.g., FH1, FIG. 5)comprising a 5′ region that is not complementary to the template nucleicacid, a downstream second probe (e.g., a fourth oligonucleotide, e.g.,FH2, FIG. 5), also comprising a 5′ region that is not complementary tothe template nucleic acid and an upstream primer (e.g., the releasedflap of a second oligonucleotide, e.g., F, FIG. 5) located not more than1000 nucleotides from the downstream first probe, with a suitable buffer(for example Sentinel Molecular Beacon PCR core buffer (Catalog #600500)or 10×Pfu buffer available from Stratagene (Catalog #200536), underconditions that allow the template nucleic acid sequence to hybridize tothe oligonucleotides (for example 95° C. for 2-5 minutes followed bycooling to between approximately 50-60° C.) to form a second duplexaccording to the invention, wherein the 5′ regions of the third andfourth probes are flaps and the extension regions of the templatenucleic acid do not form hybrids with the respective flaps. The optimaltemperature will vary depending on the specific probe(s), primers andpolymerases. The 3′ end of the upstream primer is extended by a nucleicacid polymerization activity according to the invention such that thenewly synthesized 3′ end of the upstream oligonucleotide primer isadjacent to, as defined herein, the flap of the first downstream probe.Extension is preferably carried out in the presence of 1× SentinelMolecular core buffer or 1×Pfu buffer for 15 seconds at 72° C.

The second cleavage structure is cleaved to release the flap of thefirst downstream probe (for example F, FIG. 5, step 2).

A third cleavage structure is prepared by extending the 3′ end of theupstream primer by a nucleic acid polymerization activity such that thenewly synthesized end of the upstream oligonucleotide primer is adjacentto, as defined herein, the flap of the second downstream probe.Extension is preferably carried out in the presence of 1× SentinelMolecular core buffer or 1×Pfu buffer for 15 seconds at 72° C.

6. In another embodiment of the invention, a second cleavage structureis formed by incubating a template nucleic acid comprising first andsecond extension regions (e.g., F′G1′H1′G2′H2′, FIG. 5), a downstreamfirst probe (e.g., a third oligonucleotide, e.g., FH1, FIG. 5)comprising a 5′ region that is complementary to the template nucleicacid, a downstream second probe (e.g., a fourth oligonucleotide, e.g.FH2, FIG. 5), also comprising a 5′ region that is complementary to thetemplate nucleic acid and an upstream primer (e.g., the released flap ofa second oligonucleotide, e.g., F, FIG. 5) located not more than 1000nucleotides from the upstream first probe, with a suitable buffer (forexample Sentinel Molecular Beacon PCR core buffer (Catalog #600500) or10×Pfu buffer available from Stratagene (Catalog #200536), underconditions that allow the template nucleic acid sequence to hybridize tothe oligonucleotides (for example 95° C. for 2-5 minutes followed bycooling to between approximately 50-60° C.) to form a duplex accordingto the invention, wherein the 5′ regions of the third and fourth probesare hybridized to the template nucleic acid and the extension regions ofthe template nucleic acid do not form hybrids with the respective flaps.The optimal temperature will vary depending on the specific probe(s),primers and polymerases. The 3′ end of the upstream primer is extendedby a nucleic acid polymerization activity possessing strand displacementactivity according to the invention such that the newly synthesized 3′end of the upstream oligonucleotide primer partially displaces the 5′end of the first probe. Extension is preferably carried out in thepresence of 1× Sentinel Molecular core buffer or 1×Pfu buffer for 15seconds at 72° C. The first probe is cleaved by a cleavage means, asdescribed herein, and is released from the template nucleic acid. Thenewly synthesized 3′ end of the upstream oligonucleotide is furtherextended by a nucleic acid polymerization activity possessing stranddisplacement activity and partially displaces the 5′ end of the secondprobe to form a third cleavage structure.

In one embodiment, a duplex is formed by first hybridizing the templatenucleic acid with the first probe, the second probe or both the firstand second probes, prior to the addition of the primer.

In another embodiment, a duplex is formed by hybridizing the templatenucleic acid with the probe and primer simultaneously.

A cleavage structure as described in any one of parts 1-6, above canalso be prepared as follows. The components of a cleavage structure arehybridized at a temperature (for example 50° C., 69° C., or 72° C., thatis optimal for hybridization, and subsequent steps of polymerization andcleavage, and for a time sufficient to permit hybridization andformation of a first duplex, or a second duplex, as defined herein, forexample, 15 min-1 hour.

In certain embodiments of the invention, a first or second duplex isformed by incubating the components of a first or second duplex in thepresence of a denaturing agent (e.g., DMSO or glycerol) at aconcentration that is sufficient to permit hybridization and formationof a first or second duplex, according to the invention, as well assubsequent steps of polymerization and cleavage, described below. Aconcentration of a denaturing agent that is useful according to theinvention will vary depending on the base pair compositions of thecomponents of the duplex. A concentration of a denaturing agent that isuseful according to the invention will be determined experimentally bymethods known in the art and described herein, to be sufficient topermit hybridization of complementary nucleic acids, polymerization of aprimer (e.g., a first oligonucleotide or the released flap of a secondoligonucleotide) and cleavage of a cleavage structure to release flaps,according to the invention. In one embodiment, the denaturing agent isDMSO, used at concentration of 0 to 6%, and preferably around 1.5 to 2%for nucleic acids in the range of approximately 0.1 to 1 kb. Aconcentration of DMSO greater than 2% may be used for nucleic acidsgreater than 10 kb. Alternatively, glycerol can be used as a denaturingagent at a concentration of from 0 to 10%, and preferably 5 to 8%. Both,or even other denaturing agents, may be used in combination atconcentrations that are determined experimentally by methods known inthe art.

For an isothermal reaction according to the invention, all of the stepsof the reaction that occur after the formation of a first duplex, asdefined herein, are performed at the same temperature.

A probe having a secondary structure that changes upon binding of theprobe to the target nucleic acid sequence is used to prepare a cleavagestructure according to the invention. A probe according to the inventionhas a secondary structure as defined herein, (including a stem loop, ahairpin, an internal loop, a bulge loop, a branched structure and apseudoknot) or multiple secondary structures, cloverleaf type structuresor any three-dimensional structure, as defined hereinabove. Probesuseful for forming a cleavage structure according to the invention mayalso comprise covalently bound or non-covalently bound subunits (e.g., abi-molecular or multi-molecular probe as defined herein).

B. How to Prepare a Labeled Cleavage Structure

The invention provides for labeled cleavage structures. A labeledcleavage structure is formed as described in section A1-A6 of thesection entitled “Cleavage Structure”, above, wherein one or both of theupstream probes is labeled (either at the 5′ end or internally at a sitethat is located in the flap, as defined herein) such that cleavage ofthe cleavage structure releases a labeled flap or fragment. Methods oflabeling a nucleic acid probe or oligonucleotide are well known in theart (See, Sambrook et al., supra; Ausubel et al., supra).

Subsequently, any of several strategies may be employed to distinguishthe uncleaved labeled nucleic acid from the cleaved fragments thereof.According to one embodiment, the invention provides for methods fordetecting the amount of cleaved, released, nucleic acid fragment that iscaptured by binding of a binding moiety or a tag to a capture element,respectively, on a solid support. In this manner, the present inventionpermits identification of those samples that contain a target nucleicacid sequence.

The oligonucleotide probe may be labeled, as described below, byincorporating moieties detectable by spectroscopic, photochemical,biochemical, immunochemical, enzymatic or chemical means. The method oflinking or conjugating the label to the oligonucleotide probe depends,of course, on the type of label(s) used and the position of the label onthe probe. Preferably a probe is labeled at the 5′ end although probeslabeled at the 3′ end or labeled throughout the length of the probe arealso useful in particular embodiments of the invention.

A variety of labels that would be appropriate for use in the invention,as well as methods for their inclusion in the probe, are known in theart and include, but are not limited to, enzymes (e.g., alkalinephosphatase and horseradish peroxidase) and enzyme substrates,radioactive atoms, fluorescent dyes, chromophores, chemiluminescentlabels, electrochemiluminescent labels, such as Origen™ (Igen), that mayinteract with each other to enhance, alter, or diminish a signal. Ofcourse, if a labeled molecule is used in a PCR based assay carried outusing a thermal cycler instrument, the label must be able to survive thetemperature cycling required in this automated process.

1. Radioactive Labels

Among radioactive atoms, ³³P or, ³²P is preferred. Methods forintroducing ³³P or, ³²P into nucleic acids are known in the art, andinclude, for example, 5′ labeling with a kinase, or random insertion bynick translation. “Specific binding partner” refers to a protein capableof binding a ligand molecule with high specificity, as for example inthe case of an antigen and a monoclonal antibody specific therefor.Other specific binding partners include biotin and avidin orstreptavidin, IgG and protein A, and the numerous receptor-ligandcouples known in the art. The above description is not meant tocategorize the various labels into distinct classes, as the same labelmay serve in several different modes. For example, ¹²⁵I, may serve as aradioactive label or as an electron-dense reagent. HRP may serve as anenzyme or as antigen for a monoclonal antibody. Further, one may combinevarious labels for desired effect. For example, one might label a probewith biotin, and detect the presence of the probe with avidin labeledwith ¹²⁵I, or with an anti-biotin monoclonal antibody labeled with HRP.Other permutations and possibilities will be readily apparent to thoseof ordinary skill in the art and are considered as equivalents withinthe scope of the instant invention.

2. Fluorophores

Fluorophores for use as labels in constructing labeled probes of theinvention include rhodamine and derivatives (such as Texas Red),fluorescein and derivatives (such as 5-bromomethyl fluorescein), LuciferYellow, IAEDANS, 7-Me₂N-coumarin-4-acetate,7-OH-4-CH₃-coumarin-3-acetate, 7-NH₂-4-CH₃-coumarin-3-acetate (AMCA),monobromobimane, pyrene trisulfonates, such as Cascade Blue, andmonobromorimethyl-ammoniobimane. In general, fluorophores with wideStokes shifts are preferred, to allow using fluorimeters with filtersrather than a monochromometer and to increase the efficiency ofdetection.

Probes labeled with fluorophores can readily be used in nuclease (e.g.FEN-nuclease) mediated cleavage of a cleavage structure comprising alabeled probe according to the invention. If the label is on the 5′-endof the probe, the nuclease (e.g. FEN-nuclease) generated labeledfragment is separated from the intact, hybridized probe by procedureswell known in the art. In another embodiment of the invention, detectionof the hydrolyzed, labeled probe can be accomplished using, for example,fluorescence polarization, a technique to differentiate between largeand small molecules based on molecular tumbling. Large molecules (i.e.,intact labeled probe) tumble in solution much more slowly than smallmolecules. Upon linkage of a fluorescent moiety to an appropriate siteon the molecule of interest (e.g., the 5′ end of a labeled probe), thisfluorescent moiety can be measured (and differentiated) based onmolecular tumbling, thus differentiating between intact and digestedprobe.

3. FRET

As used herein, the phrase “interactive pair of labels” as well as thephrase “pair of interactive labels” as well as the phrase “first andsecond moieties” refer to a pair of molecules which interact physically,optically, or otherwise in such a manner as to permit detection of theirproximity by means of a detectable signal. Examples of a “pair ofinteractive labels” include, but are not limited to, labels suitable foruse in fluorescence resonance energy transfer (FRET) (Stryer, L. Ann.Rev. Biochem. 47, 819-846, 1978), scintillation proximity assays (SPA)(Hart and Greenwald, Molecular Immunology 16:265-267, 1979; U.S. Pat.No. 4,658,649), luminescence resonance energy transfer (LRET) (Mathis,G. Clin. Chem. 41, 1391-1397, 1995), direct quenching (Tyagi et al.,Nature Biotechnology 16, 49-53, 1998), chemiluminescence energy transfer(CRET) (Campbell, A. K., and Patel, A. Biochem. J. 216, 185-194, 1983),bioluminescence resonance energy transfer (BRET) (Xu, Y., Piston D. W.,Johnson, Proc. Natl. Acad. Sc., 96, 151-156, 1999), or excimer formation(Lakowicz, J. R. Principles of Fluorescence Spectroscopy, KluwerAcademic/Plenum Press, New York, 1999).

The first and second moieties are “operatively coupled” to theoligonucleotides of the invention. This means that each moiety iscoupled to its respective sequence in any manner consistent with itsoperation, i.e., in any manner consistent with providing a detectablesignal upon cleavage of the probe. For example, the pair of moieties canbe either covalently or non-covalently attached to the oligonucleotidesof the invention. Examples of non-covalent attachment, any of which canbe employed to operatively couple the first or second moiety to theoligonucleotides of the invention, include attachment via hybridization,hydrogen bonds, ionic bonds, hydrophobic interactions, van der Waalsinteractions, and protein-nucleic acid interactions. Preferred aremoieties which are covalently attached at or near the 5′ and 3′ ends ofthe oligonucleotides.

As used herein, references to “fluorescence” or “fluorescent groups” or“fluorophores” include luminescence and luminescent groups,respectively.

An “increase in fluorescence”, as used herein, refers to an increase indetectable fluorescence emitted by a fluorophore. An increase influorescence may result, for example, when the distance between afluorophore and a quencher is increased, for example due to a cleavagereaction by a cleavage means, such that the quenching is reduced. Thereis an “increase in fluorescence” when the fluorescence emitted by thefluorophore is increased by at least 2 fold, for example 2, 2.5, 3, 4,5, 6, 7, 8, 10 fold or more.

a. FRET Compatible Fluorophores

A pair of interactive labels useful for the invention can comprise apair of FRET-compatible dyes, or a quencher-dye pair. In one embodiment,the pair comprises a fluorophore-quencher pair.

Oligonucleotides of the present invention permit detection/measurementof a target nucleic acid by fluorescence. They can be labeled with afluorophore and quencher in such a manner that the fluorescence emittedby the fluorophore in intact oligonucleotides is substantially quenched,whereas the fluorescence in cleaved or in some embodiments targethybridized oligonucleotides are not quenched, resulting in an increasein overall fluorescence upon probe cleavage or in some embodimentstarget hybridization. Furthermore, the generation of a fluorescentsignal during real-time detection of an amplification reaction productallows accurate quantitation of the initial number of target sequencesin a sample.

A wide variety of fluorophores can be used, including but not limitedto: 5-FAM (also called 5-carboxyfluorescein; also calledSpiro(isobenzofuran-1(3H), 9′-(9H)xanthene)-5-carboxylic acid,3′,6′-dihydroxy-3-oxo-6-carboxyfluorescein); 5-Hexachloro-Fluorescein([4,7,2′,4′,5′,7′-hexachloro-(3′,6′-dipivaloyl-fluoresceinyl)-6-carboxylicacid]); 6-Hexachloro-Fluorescein([4,7,2′,4′,5′,7′-hexachloro-(3′,6′-dipivaloylfluoresceinyl)-5-carboxylicacid]); 5-Tetrachloro-Fluorescein([4,7,2′,7′-tetra-chloro-(3′,6′-dipivaloylfluoresceinyl)-5-carboxylicacid]); 6-Tetrachloro-Fluorescein([4,7,2′,7′-tetrachloro-(3′,6′-dipivaloylfluoresceinyl)-6-carboxylicacid]); 5-TAMRA (5-carboxytetramethylrhodamine; Xanthylium,9-(2,4-dicarboxyphenyl)-3,6-bis(dimethyl-amino); 6-TAMRA(6-carboxytetramethylrhodamine; Xanthylium,9-(2,5-dicarboxyphenyl)-3,6-bis(dimethylamino); EDANS(5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid); 1,5-IAEDANS(5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid);DABCYL (4-((4-(dimethylamino)phenyl) azo)benzoic acid) Cy5(Indodicarbocyanine-5) Cy3 (Indo-dicarbocyanine-3); and BODIPY FL(2,6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-proprionicacid), Quasar-670 (Bioreseach Technologies), CalOrange (BioresearchTechnologies), Rox, as well as suitable derivatives thereof.

b. FRET Compatible Quenchers

As used herein, the term “quencher” refers to a chromophoric molecule orpart of a compound, which is capable of reducing the emission from afluorescent donor when attached to or in proximity to the donor.Quenching may occur by any of several mechanisms including fluorescenceresonance energy transfer, photoinduced electron transfer, paramagneticenhancement of intersystem crossing, Dexter exchange coupling, andexciton coupling such as the formation of dark complexes. Fluorescenceis “quenched” when the fluorescence emitted by the fluorophore isreduced as compared with the fluorescence in the absence of the quencherby at least 10%, for example, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 95%, 98%, 99%, 99.9% or more.

The quencher can be any material that can quench at least onefluorescence emission from an excited fluorophore being used in theassay. There is a great deal of practical guidance available in theliterature for selecting appropriate reporter-quencher pairs forparticular probes, as exemplified by the following references: Clegg(1993, Proc. Natl. Acad. Sci., 90:2994-2998); Wu et al. (1994, Anal.Biochem., 218:1-13); Pesce et al., editors, Fluorescence Spectroscopy(1971, Marcel Dekker, New York); White et al., Fluorescence Analysis: APractical Approach (1970, Marcel Dekker, New York); and the like. Theliterature also includes references providing exhaustive lists offluorescent and chromogenic molecules and their relevant opticalproperties for choosing reporter-quencher pairs, e.g., Berlman, Handbookof Fluorescence Spectra of Aromatic Molecules, 2nd Edition (1971,Academic Press, New York); Griffiths, Colour and Constitution of OrganicMolecules (1976, Academic Press, New York); Bishop, editor, Indicators(1972, Pergamon Press, Oxford); Haugland, Handbook of Fluorescent Probesand Research Chemicals (1992 Molecular Probes, Eugene) Pringsheim,Fluorescence and Phosphorescence (1949, Interscience Publishers, NewYork), all of which incorporated hereby by reference. Further, there isextensive guidance in the literature for derivatizing reporter andquencher molecules for covalent attachment via common reactive groupsthat can be added to an oligonucleotide, as exemplified by the followingreferences, see, for example, Haugland (cited above); Ullman et al.,U.S. Pat. No. 3,996,345; Khanna et al., U.S. Pat. No. 4,351,760, all ofwhich hereby incorporated by reference.

A number of commercially available quenchers are known in the art, andinclude but are not limited to DABCYL, BHQ-1, BHQ-2, and BHQ-3. The BHQ(“Black Hole Quenchers”) quenchers are a new class of dark quenchersthat prevent fluorescence until a hybridization event occurs. Inaddition, these new quenchers have no native fluorescence, virtuallyeliminating background problems seen with other quenchers. BHQ quencherscan be used to quench almost all reporter dyes and are commerciallyavailable, for example, from Biosearch Technologies, Inc (Novato,Calif.).

c. Attachment of Fluorophore and Quencher

In one embodiment of the invention, the fluorophore or quencher isattached to the 3′ nucleotide of an oligonucleotide of the invention. Inanother embodiment of the invention, the fluorophore or quencher isattached to the 5′ nucleotide. In yet another embodiment, thefluorophore or quencher is internally attached to an oligonucleotide ofthe invention. In some embodiments, either the fluorophore or quencheris attached to the 5′ nucleotide of the probe and the other of saidfluorophore or quencher is attached to the 3′ nucleotide of the probe.In other embodiments, either the fluorophore or quencher is attached toa probe of the invention and the other of said fluorophore or quencheris attached to a template nucleic acid of the invention. Attachment canbe made via direct coupling, or alternatively using a spacer moleculeof, for example, from about 1 to about 5 atoms in length.

For the internal attachment of the fluorophore or quencher, linkage canbe made using any of the means known in the art. Appropriate linkingmethodologies for attachment of many dyes to oligonucleotides aredescribed in many references, e.g., Marshall, Histochemical J., 7:299-303 (1975); Menchen et al., U.S. Pat. No. 5,188,934; Menchen et al.,European Patent Application 87310256.0; and Bergot et al., InternationalApplication PCT/US90/05565. All are hereby incorporated by reference.

Each member of the fluorophore/quencher pair can be attached anywherewithin the oligonucleotide, preferably at a distance from the other ofthe pair such that sufficient amount of quenching occurs when theoligonucleotides are not cleaved or is hybridized to the templatenucleic acid.

In one embodiment, one can use two interactive labels (e.g., FRET ornon-FRET pairs) on a single oligonucleotide probe with due considerationgiven for maintaining an appropriate spacing of the labels on theoligonucleotide to permit the separation of the labels duringoligonucleotide probe unfolding (e.g., for example due to a change inthe secondary structure of the probe) or hydrolysis. In anotherembodiment, one can use two olgionucltoitides of the invention, e.g.,template nucleic acid and third oligonucleotide, each with one member ofa pair of interactive labels.

In certain embodiments, the fluorescence of the released label is thencompared to the label remaining bound to the target. It is not necessaryto separate the nuclease (e.g. FEN-nuclease) generated fragment and theprobe that remains bound to the target after cleavage in the presence ofnuclease (e.g. FEN-nuclease) if the probe is synthesized with afluorophore and a quencher that are separated by about 20 nucleotides.Alternatively, the quencher is positioned such that the probe will notfluoresce when not hybridized to the target nucleic acid sequence. Sucha dual labeled probe will not fluoresce when intact or when nothybridized to the target nucleic acid sequence (or in the case of bi- ormultimolecular probes, when the probe is not dissociated) because thelight emitted from the dye is quenched by the quencher. Thus, anyfluorescence emitted by an intact probe is considered to be backgroundfluorescence. In one embodiment, when a labeled probe is cleaved by aFEN nuclease, dye and quencher are separated and the released fragmentwill fluoresce. Alternatively, when a labeled probe is hybridized to atarget nucleic acid, the distance between the dye and the quencher isincreased and the level of fluorescence increases. In an embodimentwherein the probe is a bi- or multi-molecular probe, dissociation of themolecules comprising the probe results in an increase in fluorescence.The amount of fluorescence is proportional to the amount of nucleic acidtarget sequence present in a sample.

In yet another embodiment, two labeled nucleic acids are used, eachcomplementary to separate regions of separate strands of adouble-stranded target sequence, but not to each other, so that alabeled nucleic acid anneals downstream of each primer. For example, thepresence of two probes can potentially double the intensity of thesignal generated from a single label and may further serve to reduceproduct strand reannealing, as often occurs during PCR amplification.The probes are selected so that the probes bind at positions adjacent(downstream) to the positions at which primers bind.

In still another embodiment, two labeled nucleic acids are used, eachcomplementary to at least a portion of each other, e.g., templatenucleic acid and third oligonucleotide. For example, the templatenucleic acid may be labeled with a quencher and the thirdoligonucleotide may be labeled with a fluorescer. When hybridized toeach other the fluorescer is substantially quenched. However, uponcleavage of the third oligonucleotide the labels are separated andfluorescence substantially increases.

One can also use multiple probes in the present invention to achieveother benefits. For instance, one could test for any number of pathogensin a sample simply by putting as many probes as desired into thereaction mixture; the probes could each comprise a different label tofacilitate detection. For example, multiple sets of different secondoligonucleotides, template nucleic acids and third oligonucleotides canbe added to a reaction mixture. Each of the second oligonucleotideswould be complementary to a region of a nucleic acid sequence from adifferent pathogen. If the pathogen is present in the reaction mixturethe corresponding second oligonucleotide would hybridize to thepathogens nucleic acid sequence. Upon polymerization of the firstoligonucleotide the second oligonucleotide would be cleaved. The cleavedflap would then hybridize to a template nucleic acid specific for thisflap. The flap would be extended by a nucleic acid polymerase forming asecond cleavage structure with the third oligonucleotide, which iscleaved. Cleavage of the third oligonucleotide would produce a signalindicative of the particular pathogen. Multiple different signals, e.g.,fluorescent signals, could be generated for each pathogen of interest.Such reactions can be conducted in the Mx3000P Real Time PCR system(Stratagene, Calif.).

One can also achieve allele-specific or species-specific discriminationusing multiple probes in the present invention, for instance, by usingprobes that have different T_(m)s and conducting the annealing/cleavagereaction at a temperature specific for only one probe/allele duplex. Onecan also achieve allele specific discrimination by using only a singleprobe and examining the types of cleavage products generated. In oneembodiment of the invention, the probe is designed to be exactlycomplementary, at least in the 5′ terminal region, to one allele but notto the other allele(s). With respect to the other allele(s), the probewill be mismatched in the 5′ terminal region of the probe so that adifferent cleavage product will be generated as compared to the cleavageproduct generated when the probe is hybridized to the exactlycomplementary allele.

Although probe sequence can be selected to achieve important benefits,one can also realize important advantages by selection of probelabels(s). The labels may be attached to the oligonucleotide directly orindirectly by a variety of techniques. Depending on the precise type oflabel used, the label can be located at the 5′ or 3′ end of the probe,located internally in the probe, or attached to spacer arms of varioussizes and compositions to facilitate signal interactions. Usingcommercially available phosphoramidite reagents, one can produceoligomers containing functional groups (e.g., thiols or primary amines)at either the 5′ or the 3′ terminus via an appropriately protectedphosphoramidite, and can label them using protocols described in, forexample, PCR Protocols: A Guide to Methods and Applications, Innis etal., eds. Academic Press, Ind., 1990.

Methods for introducing oligonucleotide functionalizing reagents tointroduce one or more sulfhydryl, amino or hydroxyl moieties into theoligonucleotide probe sequence, typically at the 5′ terminus, aredescribed in U.S. Pat. No. 4,914,210. A 5′ phosphate group can beintroduced as a radioisotope by using polynucleotide kinase andgamma-³²P-ATP or gamma-³³P-ATP to provide a reporter group. Biotin canbe added to the 5′ end by reacting an aminothymidine residue, or a6-amino hexyl residue, introduced during synthesis, with anN-hydroxysuccinimide ester of biotin. Labels at the 3′ terminus mayemploy polynucleotide terminal transferase to add the desired moiety,such as for example, cordycepin ³⁵S-dATP, and biotinylated dUTP.

Oligonucleotide derivatives are also available labels. For example,etheno-dA and etheno-A are known fluorescent adenine nucleotides thatcan be incorporated into a nucleic acid probe. Similarly, etheno-dC or2-amino purine deoxyriboside is another analog that could be used inprobe synthesis. The probes containing such nucleotide derivatives maybe hydrolyzed to release much more strongly fluorescent mononucleotidesby flap-specific nuclease activity.

A labeled probe having a secondary structure that changes upon bindingof the probe to the target nucleic acid sequence is used to prepare alabeled cleavage structure according to the invention. A labeled probeaccording to the invention has a secondary structure as defined herein,(including a stem loop, a hairpin, an internal loop, a bulge loop, abranched structure and a pseudoknot) or multiple secondary structures,cloverleaf type structures or any three-dimensional structure, asdefined hereinabove. Labeled probes useful for forming a labeledcleavage structure according to the invention may also comprisecovalently bound or non-covalently bound subunits (e.g., a bi-molecularor multi-molecular probe as defined herein).

C. Cleaving a Cleavage Structure and Generating a Signal

A cleavage structure according to the invention can be cleaved by themethods described in the section above, entitled “Nucleases”.

D. Detection of Released Labeled Fragments

Detection or verification of the labeled fragments may be accomplishedby a variety of methods well known in the art and may be dependent onthe characteristics of the labeled moiety or moieties comprising alabeled cleavage structure.

V. Cleavage Means

Nucleases useful according to the invention include any enzyme thatpossesses 5′ endonucleolytic activity for example a DNA polymerase, e.g.DNA polymerase I from E. coli, and DNA polymerase from Thermus aquaticus(Taq), Thermus thermophilus (Tth), and Thermus flavus (Tfl). Nucleasesuseful according to the invention also include DNA polymerases with5′-3′ exonuclease activity, including but not limited to eubacterial DNApolymerase I, including enzymes derived from Thermus species (Taq, Tfl,Tth, Tca (caldophilus) Tbr (brockianus), enzymes derived from Bacillusspecies (Bst, Bca, Magenta (full length polymerases, NOT N-truncatedversions)), enzymes derived from Thermotoga species (Tma (maritima, Tne(neopolitana) and E. coli DNA polymerase I. The term nuclease alsoembodies FEN nucleases.

FEN-1 is an ˜40 kDa divalent metal ion-dependent exo- and endonucleasethat specifically recognizes the backbone of a 5′ single-stranded flapstrand and tracks down this arm to the cleavage site, which is locatedat the junction wherein the two strands of duplex DNA adjoin the flap.Both the endo- and exonucleolytic activities show little sensitivity tothe base at the most 5′ position at the flap or nick. Both FEN-1 endo-and exonucleolytic substrate binding and cutting are stimulated by anupstream oligonucleotide (flap adjacent strand or primer). This is alsothe case for E. coli pol I. The endonuclease activity of the enzyme isindependent of the 5′ flap length, cleaving a 5′ flap as small as onenucleotide. The endonuclease and exonuclease activities are insensitiveto the chemical nature of the substrate, cleaving both DNA and RNA.

Both the endo- and exonucleolytic activities are inhibited byconcentrations of salts in the physiological range. The exonucleaseactivity is inhibited 50-fold at 50 mM NaCl as compared to 0 mM NaCl.The endonuclease activity is inhibited only sevenfold at 50 mM NaCl(Reviewed in Lieber 1997, supra).

Although a 5′-OH terminus is a good substrate for FEN-1 loading onto a5′ flap substrate, it serves as a very poor substrate when part of anick in an otherwise double stranded DNA structure. The electrostaticrepulsion by the terminal phosphate is likely to favor breathing of thesubstrate into a pseudo-flap configuration, providing the active form ofthe substrate for FEN-1. Such an explanation would indicate a singleactive site and a single mechanism of loading of FEN-1 onto the 5′ ssDNAterminus of the flap or pseudo-flap configuration of the nick.Consistent with this model are observations that optimal activity at anick requires very low Mg²⁺ and monovalent salt concentrations, whichdestabilize base-pairing and would favor breathing of a nick to a flap.Higher Mg²⁺ and monovalent salt concentrations would disfavor breathingand inhibit cutting of nicked or gapped structures that do requirebreathing to convert to a flap. Cleavage of stable flap structures isoptimal at moderate Mg²⁺ levels and does not decrease with increasingMg²⁺ concentration. This is because a flap substrate does not have tomelt out base pairs to achieve its structure; hence, it is entirelyinsensitive to Mg²⁺. Though the endonucleolytic activity decreases withmonovalent salt, the decline is not nearly as sharp as that seen for theexonucleolytic activity. Furthermore, it has previously been shown thatone-nucleotide flaps are efficient substrates. All of these observationsare consistent with the fact that when FEN-1 has been interpreted to befunctioning as an exonuclease, the size of the degradation products varyfrom one to several nucleotides in length. Breathing of nicks into flapsof varying length would be expected to vary with local sequence,depending on the G/C content. In summary, a nick breathing to form atransient flap means that the exonucleolytic activity of FEN-1 is thesame as the endonucleolytic activity (Reviewed in Lieber, 1997, supra).

The endonuclease and exonuclease activities of FEN-1 cleave both DNA andRNA without requiring accessory proteins. At the replication fork,however, FEN-1 does interact with other proteins, including a DNAhelicase and the proliferating cell nuclear antigen (PCNA), theprocessivity factor for DNA polymerases δ and ε. PCNA significantlystimulates FEN-1 endo- and exonucleolytic activity.

The FEN-1 enzymes are functionally related to several smallerbacteriophage 5′→3′ exonucleases such as T5 5′ exonuclease and T4 RNaseH as well as to the larger eukaryotic nucleotide excision repair enzymessuch as XPG, which also acts in the transcription-coupled repair ofoxidative base damage. In eubacteria such as Escherichia coli andThermus aquaticus, Okazaki processing is provided by the PolI 5′→3′exonuclease domain. These bacterial and phage enzymes share two areas oflimited sequence homology with FEN-1, which are termed the N(N-terminal)and I (intermediate) regions, with the residue similarities concentratedaround seven conserved acidic residues. Based on crystal structures ofT4 RNase H and T5 exonuclease as well as mutagenesis data, it has beenproposed that these residues bind to two Mg²⁺ ions that are required foraffecting DNA hydrolysis; however, the role each metal plays in thecatalytic cycle, which is subtly different for each enzyme, is not wellunderstood (Reviewed in Hosfield et al., 1998b, supra).

FEN-1 genes encoding FEN-1 enzymes useful in the invention includemurine fen-1, human fen-1, rat fen-1, Xenopus laevis fen-1, and fen-1genes derived from four archaebacteria Archaeglobus fulgidus,Methanococcus jannaschii, Pyrococcus furiosus and Pyrococcus horikoshii.cDNA clones encoding FEN-1 enzymes have been isolated from human(GenBank Accession Nos.: NM_(—)004111 and L37374), mouse (GenBankAccession No.: L26320), rat (GenBank Accession No.: AA819793), Xenopuslaevis (GenBank Accession Nos.: U68141 and U64563), and P. furiosus(GenBank Accession No.: APF03497). The complete nucleotide sequence forP. horikoshii flap endonuclease has also been determined (GenBankAccession No.: AB005215). The FEN-1 family also includes theSaccharomyces cerevisiae RAD27 gene (GenBank Accession No.: Z28113Y13137) and the Saccharomyces pombe RAD2 gene (GenBank Accession No.:X77041). The archaeal genome of Methanobacterium thermautotrophiculumhas also been sequenced. Although the sequence similarity between FEN-1and prokaryotic and viral 5′→3′ exonucleases is low, FEN-1s within theeukaryotic kingdom are highly conserved at the amino acid level, withthe human and S. cerevisiae proteins being 60% identical and 78%similar. The three archaebacterial FEN-1 proteins are also, highlyhomologous to the eukaryotic FEN-1 enzymes (Reviewed in Matsui et al.,1999., J. Biol. Chem., 274:18297, Hosfield et al., 1998b, J. Biol.Chem., 273:27154 and Lieber, 1997, BioEssays, 19:233).

The sequence similarities in the two conserved nuclease domains(N-terminal or N and intermediate or I domains) between human and otherFEN-1 family members are 92% (murine), 79% (S. cerevisiae), 77% (S.pombe), 72% (A. fulgidus), 76% (M. jannaschii), and 74% (P. furiosus).

FEN-1 specifically recognizes the backbone of a 5′ single-stranded flapstrand and migrates down this flap arm to the cleavage site located atthe junction between the two strands of duplex DNA and the flap. If thestrand upstream of the flap (sometimes called the flap adjacent strandor primer strand) is removed, the resulting structure is termed apseudo-Y (see FIG. 8). This structure is cleaved by FEN-1, but at 20- to100-fold lower efficiency. FEN-1 does not cleave 3′ single-strandedflaps. However, FEN-1 acting as an exonuclease will hydrolyze dsDNAsubstrates containing a gap or nick (Reviewed in Hosfield et al., 1998a,supra, Hosfield et al., 1999b, supra, and Lieber 1997, supra).Exonucleolytically, FEN-1 acts at a nick and, with lower efficiency, ata gap or a recessed 5′ end on dsDNA. At gapped structures, theefficiency of FEN-1 binding and cutting decreases with increasing gapsize up to approximately five nucleotides and then stabilizes at a levelof cleavage that is equivalent to activity on a recessed 5′ end withindsDNA. Blunt dsDNA, recessed 3′ ends and ssDNA are not cleaved (Reviewedin Lieber 1997, supra).

FEN nucleases that are useful according to the invention have beenisolated from a variety of organisms including human (GenBank AccessionNos.: NM_(—)004111 and L37374), mouse (GenBank Accession No.: L26320),rat (GenBank Accession No.: AA819793), yeast (GenBank Accession No.:Z28113 Y13137 and GenBank Accession No.: X77041) and xenopus laevis(GenBank Accession Nos.: U68141 and U64563). Such enzymes can be clonedand overexpressed using conventional techniques well known in the art.

A FEN nuclease according to the invention is preferably thermostable.Thermostable FEN nucleases have been isolated and characterized from avariety of thermostable organisms including four archeaebacteria. ThecDNA sequence (GenBank Accession No.: AF013497) and the amino acidsequence (Hosfield et al., 1998a, supra and Hosfield et al., 1998b) forP. furiosus flap endonuclease have been determined. The completenucleotide sequence (GenBank Accession No.: AB005215) and the amino acidsequence (Matsui et al., supra) for P. horikoshii flap endonuclease havealso been determined. The amino acid sequence for M jannaschii (Hosfieldet al., 1998b and Matsui et al., 1999 supra) and A. fulgidus (Hosfieldet al., 1998b) flap endonuclease have also been determined.

Thermostable FEN1 enzymes can be cloned and overexpressed usingtechniques well known in the art and described in Hosfield et al.,1998a, supra, Hosfield et al., 1998b, Kaiser et al., 1999, J. Biol.Chem., 274: 21387 and Matusi et al., supra and herein in Example 5entitled “Cloning Pfu FEN-1”.

The endonuclease activity of a FEN enzyme can be measured by a varietyof methods including the following.

A. FEN EENDONUCLEASE ACTIVITY ASSAY

1. Templates (for example as shown in FIG. 14) are used to evaluate theactivity of a FEN nuclease according to the invention.

Template 1 is a 5′³³P labeled oligonucleotide (Heltest4) with thefollowing sequence: 5′AAAATAAATAAAAAAAATACTGTTGGGAAGGGCGATCGGTGCG3′. Theunderlined section of Heltest4 represents the region complementary toM13mp18+. The cleavage product is an 18 nucleotide fragment with thesequence AAAATAAATAAAAAAAAT.

Heltest4 binds to M13 to produce a complementary double stranded domainas well as a non-complementary 5′ overhang. This duplex forms template 2(FIG. 14) which is also used for helicase assays. Template 3 (FIG. 14)has an additional primer (FENAS) bound to M13 and is directly adjacentto Heltest 4. The sequence of FENAS is: 5′CCATTCGCCATTCAGGCTGCGCA3′. Inthe presence of template 3, FEN binds the free 5′ terminus of Heltest4,migrates to the junction and cleaves Heltest4 to produce an 18nucleotide fragment. Templates 1 and 2 serve as controls, althoughtemplate 2 can also serve as a template.

Templates are prepared as described below:

Template 1 Template 2 Template 3 Heltest4 14 μl 14 μl 14 μl M13 ** 14 μl14 μl FENAS ** ** 14 μl H₂O 28 μl 14 μl ** 10x Pfu Buff. 4.6 μl  4.6 μl 4.6 μl 

10×Pfu buffer is available from Stratagene (Catalog # 200536). Accordingto the method of the invention, 10×Pfu buffer is diluted such that areaction is carried out in the presence of 1× buffer.

M13 is M13mp18+strand and is at a concentration of 200 ng/μL, ³³Plabeled Heltest4 is at an approximate concentration of 0.7 ng/μl, andFENAS is at a concentration of 4.3 ng/μl. Based on these concentrations,the Heltest4 and M13 are at approximately equal molar amounts (5×10⁻¹⁴)and FENAS is present in an approximately 10× molar excess (6×10⁻¹³).

The template mixture is heated at 95° C. for five minutes, cooled toroom temperature for 45 minutes and stored at 4° C. overnight.

2 μl of FEN-1 or, as a control, H₂O are mixed with the three templatesas follows:

3 μl template 0.7 μl 10x cloned Pfu buffer 0.56 μl 100 mM MgCl₂ 2.00 μlenzyme or H₂O 0.74 μl H₂0 7.00 μl total volume

The reactions are allowed to proceed for 30 minutes at 50° C. andstopped by the addition of 2 μl formamide “Sequencing Stop” solution toeach sample. Samples are heated at 95° C. for five minutes and loaded ona 6% acrylamide, 7M urea CastAway (Stratagene) gel.

Alternatively, FEN activity can be analyzed in the following bufferwherein a one hour incubation time is utilized.

10×FEN Buffer

500 mM Tris-HCl pH 8.0

100 mM MgCl₂

The reaction mixture below is mixed with 2 μl of FEN or, as a control, 2μl of H₂O.

3 μl template 0.7 μl 10x FEN buffer 2.00 μl enzyme or H₂O 1.3 μl H₂O7.00 μl total volume

Samples are incubated for one hour at 50° C. in a Robocyler 96 hot topthermal cycler. Following the addition of 2 μl of Sequencing Stop dyesolution, samples are heated at 99° C. for five minutes. Samples areloaded on an eleven-inch long, hand-poured, 20% acrylamide/bisacrylamide, 7M urea gel. The gel is run at 20 watts until thebromophenol blue has migrated approximately ⅔ the total distance. Thegel is removed from the glass plates and soaked for 10 minutes in fix(15% methanol, 5% acetic acid) and then for 10 minutes in water. The gelis placed on Whatmann 3 mm paper, covered with plastic wrap and driedfor 2 hours in a heated vacuum gel dryer. The gel is exposed overnightto X-ray film.

2. FEN endonuclease activity can also be measured according to themethod of Kaiser et al., supra). Briefly, reactions are carried out in a10 μl volume containing 10 mM MOPS, pH 7.5, 0.05% Tween 20, 0.05%Nonidet P-40, 10 1 g/ml tRNA, and 200 mM KCl for TaqPol and TthPol or 50mM KCl for all other enzymes. Reaction conditions can be varieddepending on the cleavage structure being analyzed. Substrates (2 μM)and varying amounts of enzyme are mixed with the indicated (above)reaction buffer and overlaid with Chill-out (MJ Research) liquid wax.Substrates are heat denatured at 90° C. for 20 s and cooled to 50° C.,then reactions are started by addition of MgCl₂ or MnCl₂ and incubatedat 50° C. for the specified length of time. Reactions are stopped by theaddition of 1011 of 95% formamide containing 10 mM EDTA and 0.02% methylviolet (Sigma). Samples are heated to 90° C. for 1 min immediatelybefore electrophoresis on a 20% denaturing acrylamide gel (19:1cross-linked), with 7M urea, and in a buffer of 45 mM Tris borate, pH8.3, 1.4 mM EDTA. Unless otherwise indicated, 1 μl of each stoppedreaction is loaded per lane. Gels are scanned on an FMBIO-100fluorescent gel scanner (Hitachi) using a 505-nm filter. The fraction ofcleaved product is determined from intensities of bands corresponding touncut and cut substrate with FMBIO Analysis software (version 6.0,Hitachi). The fraction of cut product should not exceed 20% to ensurethat measurements approximate initial cleavage rates. The cleavage rateis defined as the concentration of cut product divided by the enzymeconcentration and the time of the reaction (in minutes). For each enzymethree data points are used to determine the rate and experimental error.

3. FEN endonuclease activity can also be measured according to themethod of Hosfield et al., 1998a, supra. Briefly, in a final volume of13 μl, varying amounts of FEN and 1.54 pmol of labeled cleavagesubstrate are incubated at different temperatures for 30 min before thereaction is quenched with an equal volume of stop solution (10 mM EDTA,95% deionized formamide, and 0.008% bromophenol blue and xylene cyanol).Samples are electrophoresed through denaturing 15% polyacrylamide gels,and the relative amounts of starting material and product arequantitated using the IPLabGel system (Stratagene) running MacBAS imageanalysis software. Most reactions are performed in standard assay buffer(10 mM Tris-HCl (pH 8.0), 10 mM MgCl₂, and 50 μg/ml bovine serumalbumin); however, in a series of experiments the effect of differentdivalent metals and pH levels are studied by varying the standardbuffer. For divalent metals, MgCl₂ is omitted, and different metal ionsare used at a final concentration of 10 mM. To study the influence ofpH, buffers containing different amounts of Tris-HCl, glycine, andsodium acetate are used at a final concentration of 10 mM to obtain awide range of pH levels at 25° C.

4. FEN endonuclease activity can also be measured according to themethod of Matusi et al., 1999, supra. Briefly, the enzyme reactions areperformed in a 15-μl reaction mixture containing 50 mM Tris-HCl (pH7.4), 1.5 mM MgCl₂, 0.5 mM β-mercaptoethanol, 100 μg/ml bovine serumalbumin, and 0.6 pmol of a labeled cleavage structure. After incubationfor 30 min at 60° C., the reaction is terminated by adding 15 μl of 95%formamide containing 10 mM EDTA and 1 mg/ml bromphenol blue. The samplesare heated at 95° C. for 10 min, loaded onto a 15% polyacrylamide gel(35 cm×42.5 cm) containing 7M urea and 10×TBE (89 mM Tris-HCl, 89 mMboric acid, 2 mM EDTA (pH 8.0)), and then electrophoresed for 2 h at2000 V. Reaction products are visualized and quantified using aPhosphorImager (Bio-Rad). Size marker, oligonucleotides are 5′end-labeled with [γ-³²P]ATP and T4 polynucleotide kinase.

To determine the optimum pH, the reaction is performed in an assaymixture (15 μl) containing 1.5 mM MgCl₂, 0.5 mM β-mercaptoethanol, 100μg/ml bovine serum albumin, and 0.6 pmol of 5′ end-labeled cleavagestructure in 50 mM of one of the following buffers at 60° C. for 30 min.Three different 50 mM buffers are used to obtain a wide pH range asfollows: sodium acetate buffer (pH 4.0-5.5), phosphate buffer (pH5.5-8.0), and borate buffer (pH 8.0-9.4).

B. FEN EXONUCLEASE ACTIVITY ASSAY

The exonuclease activity of a FEN nuclease according to the inventioncan be measured by the method of measuring FEN-1 endonuclease activitydescribed in Matsui et al., 1999, supra and summarized above.

Alternatively, the exonuclease activity of a FEN enzyme can be analyzedby the method described in Hosfield et al., 1998b, supra. Briefly,exonuclease activities are assayed using a nicked substrate of FEN underconditions identical to those described for the endonuclease assays(described above).

The precise positions of DNA cleavage in both the exonuclease andendonuclease experiments can be obtained by partial digestion of a5′³²P-labeled template strand using the 3′-5′ exonuclease activity ofKlenow fragment.

A cleavage structure according to the invention is described in thesection entitled “Cleavage Structure”.

VI. Determining the Stability of the Secondary Structure of a Probe

A. Melting Temperature Assay

A melting temperature assay, takes advantage of the different absorptionproperties of double stranded and single stranded DNA, that is, doublestranded DNA (the double stranded DNA being that portion of a nucleicacid sequence that has folded back on itself to generate an antiparallelduplex structure wherein complementary sequences (base pairs) areassociated via hydrogen bonding) absorbs less light than single strandedDNA at a wavelength of 260 nm, as determined by spectrophotometricmeasurement.

The denaturation of DNA occurs over a narrow temperature range andresults in striking changes in many of the physical properties of DNA. Aparticularly useful change occurs in optical density. The heterocyclicrings of nucleotides adsorb light strongly in the ultraviolet range(with a maximum close to 260 nm that is characteristic for each base).However, the adsorption of DNA is approximately 40% less than would bedisplayed by a mixture of free nucleotides of the same composition. Thiseffect is called hyperchromism and results from interactions between theelectron systems of the bases, made possible by their stacking in theparallel array of the double helix. Any departure from the duplex stateis immediately reflected by a decline in this effect (that is, by anincrease in optical density toward the value characteristic of freebases (FIG. 13 a). The denaturation of double stranded DNA can thereforebe followed by this hyperchromicity (FIGS. 13 b and 13 c)

The midpoint of the temperature range over which the strands of DNAseparate is called the melting temperature, denoted T_(m). An example ofa melting curve determined by change in optical absorbance is shown inFIG. 13 c. The curve always takes the same form, but its absoluteposition on the temperature scale (that is, its T_(m)) is influenced byboth the base composition of the DNA and the conditions employed fordenaturation.

The melting temperature of a DNA molecule depends markedly on its basecomposition. DNA molecules rich in GC base pairs have a higher Tm thanthose having an abundance of AT base pairs (FIG. 13 b). The Tm of DNAfrom many species varies linearly with GC content, rising from 77° to100° C. as the fraction of GC pairs increases from 20% to 78%. That is,the dependence of T_(m) on base composition is linear, increasing about0.4° C. for every percent increase in G-C content. GC base pairs aremore stable than AT pairs because their bases are held together by threehydrogen bonds rather than by two. In addition, adjacent GC base pairsinteract more strongly with one another than do adjacent AT base pairs.Hence, the AT-rich regions of DNA are the first to melt.

A major effect on T_(m) is exerted by the ionic strength of thesolution. The T_(m) increases 16.6° C. for every tenfold increase inmonovalent cation concentration. The most commonly used condition is toperform manipulations of DNA in 0.12 M phosphate buffer, which providesa monovalent Na+ concentration of 0.18M, and a T_(m) of the order of 90°C.

The T_(m) can be greatly varied by performing the reaction in thepresence of reagents, such as formamide, that destabilize hydrogenbonds. This allows the T_(m) to be reduced to as low as 40° C. with theadvantage that the DNA does not suffer damage (such as strand breakage)that can result from exposure to high temperatures. (Stryer,Biochemistry, 1998, 3^(rd) Edition, W.H. Freeman and Co., pp. 81-82 andLewin, Genes II, 1985, John Wiley & Sons, p. 63-64).

The stability of the secondary structure of the probe according to theinvention is determined in a melting temperature assay as follows.

A standard curve for the probe (for example FIG. 13 c), whereinabsorbance is plotted versus temperature, is prepared by incubating asample comprising from about 0.2 μg/ml to 100 μg/ml of the probe in abuffer which allows for denaturing and reannealing of the probe atvarious temperature and for a time sufficient to permit denaturing andreannealing of the probe, and measuring the absorbance of a sample in aquartz cuvette (with a pathlength appropriate for the spectrophotometerbeing used, e.g., 1-cm), in a spectrophotometer over a range oftemperatures wherein the lower temperature limit of the range is atleast 50° C. below, and the upper temperature limit of the range is atleast 50° C. above the Tm or predicted Tm of the probe. The Tm of theprobe is predicted based on the base pair composition according tomethods well known in the art (see, Sambrook, supra; Ausubel, supra).Standard curves are generated and compared, using a variety of buffers(e.g., 1×TNE buffer (10×-0.1M Tris base, 10 mM EDTA, 2.0 M NaCl, pH7.4), FEN nuclease buffer, described herein, 1× Cloned Pfu buffer,described herein, 1× Sentinel Molecular beacon buffer, described herein)including a buffer that is possible and preferentially optimal for theparticular nuclease to be employed in the cleavage reaction. The pH ofthe buffer will be monitored as the temperature increases, and adjustedas is needed.

The assay is performed in a single-beam ultraviolet to visible range(UV-VIS) spectrophotometer. Preferably, the assay is performed in adouble-beam spectrophotometer to simplify measurements by automaticallycomparing the cuvette holding the sample solution to a reference cuvette(matched cuvette) that contains the blank. The blank is an equal volumeof sample buffer.

The temperature of the spectrophotometer can be controlled such that theabsorbance of the sample is measured at specific temperatures.Spectrophotometers useful according to the invention include but are notlimited to the Beckman Coulter DU® 600/7000 Spectrophotometers incombination with the MicroTm Analysis Accessory (Beckman Coulter, Inc.,Columbia, Md.).

The stability of the secondary structure of a probe at a particulartemperature and in a buffer that is possible and preferentially optimalfor the nuclease to be employed in the cleavage reaction of the probe,is determined by measuring the absorbance of the probe at a particulartemperature, as above, and determining if the value of the absorbance isless than the absorbance at the Tm, as determined from the standardcurve, wherein the standard curve is generated using either the samebuffer as used at the test temperature, or a buffer known to produce acomparable standard curve, as described above. The secondary structureof the probe is “stable” in a melting temperature assay, at atemperature that is at or below the temperature of the cleavage reaction(i.e., at which cleavage is performed) if the level of light absorbanceat the temperature at or below the temperature of the cleavage reactionis less (i.e., at least 5%, preferably 20% and most preferably 25% ormore) than the level of light absorbance at a temperature that is equalto the Tm of the probe (see FIGS. 13 c and 13 d).

B. FRET

A FRET assay is useful in the invention for two purposes. The first isto determine whether the secondary structure of a probe is “stable” asdefined herein. The second is to determine whether the secondarystructure of the probe has undergone a “change” upon binding of theprobe to the target nucleic acid or whether the probe has been cleavedby a cleavage means.

“FRET” is a distance-dependent interaction between the electronicexcited states of two dye molecules in which excitation is transferredfrom a donor molecule to an acceptor molecule. FRET is caused by achange in the distance separating a fluorescent donor group from aninteracting resonance energy acceptor, either another fluorophore, achromophore, or a quencher. Combinations of donor and acceptor moietiesare known as “FRET pairs”. Efficient FRET interactions require that theabsorption and emission spectra of the dye pairs have a high degree ofoverlap.

In most embodiments, the donor and acceptor dyes for FRET are different,in which case FRET can be detected by the appearance of sensitizedfluorescence of the acceptor and/or by quenching of donor fluorescence.When the donor and acceptor are the same, FRET is detected by theresulting fluorescence depolarization. FRET is dependent on the inversesixth power of the intermolecular separation (Stryer et al., 1978, Ann.Rev. Biochem., 47:819; Selvin, 1995, Methods Enzymol., 246:300).

As used herein, the term “donor” refers to a fluorophore which absorbsat a first wavelength and emits at a second, longer wavelength. The term“acceptor” refers to a fluorophore, chromophore or quencher with anabsorption spectrum which overlaps the donor's emission spectrum and isable to absorb some or most of the emitted energy from the donor when itis near the donor group (typically between 1-100 nm). If the acceptor isa fluorophore capable of exhibiting FRET, it then re-emits at a third,still longer wavelength; if it is a chromophore or quencher, then itreleases the energy absorbed from the donor without emitting a photon.Although the acceptor's absorption spectrum overlaps the donor'semission spectrum when the two groups are in proximity, this need not bethe case for the spectra of the molecules when free in solution.Acceptors thus include fluorophores, chromophores or quenchers whichexhibit either FRET or quenching when placed in proximity, on a probeaccording to the invention, to the donor due to the presence of a probesecondary structure that changes upon binding of the probe to the targetnucleic acid, as defined herein. Acceptors do not include fluorophores,chromophores or quenchers that exhibit FRET or quenching a) attemperatures equal to or greater than the Tm (e.g. more than 5° C. abovethe Tm, for example 6° C., 10° C., 25° C., 50° C. or more above the Tm)or b) in the presence of a target nucleic acid.

Reference herein to “fluorescence” or “fluorescent groups” or“fluorophores” include luminescence, luminescent groups and suitablechromophores, respectively. Suitable luminescent probes include, but arenot limited to, the luminescent ions of europium and terbium introducedas lanthium chelates (Heyduk & Heyduk, 1997). The lanthanide ions arealso good donors for energy transfer to fluorescent groups (Selvin1995). Luminescent groups containing lanthanide ions can be incorporatedinto nucleic acids utilizing an ‘open cage’ chelator phosphoramidite.

As used herein, the term “quenching” refers to the transfer of energyfrom donor to acceptor which is associated with a reduction of theintensity of the fluorescence exhibited by the donor.

The donor and acceptor groups may independently be selected fromsuitable fluorescent groups, chromophores and quenching groups. Donorsand acceptors useful according to the invention include but are notlimited to: 5-FAM (also called 5-carboxyfluorescein; also calledSpiro(isobenzofuran-1(3H), 9′-(9H)xanthene)-5-carboxylicacid,3′,6′-dihydroxy-3-oxo-6-carboxyfluorescein);5-Hexachloro-Fluorescein([4,7,2′,4′,5′,7′-hexachloro-(3′,6′-dipivaloyl-fluoresceinyl)-6-carboxylicacid]); 6-Hexachloro-Fluorescein([4,7,2′,4′,5′,7′-hexachloro-(3′,6′-dipivaloylfluoresceinyl)-5-carboxylicacid]); 5-Tetrachloro-Fluorescein([4,7,2′,7′-tetrachloro-(3′,6′-dipivaloylfluoresceinyl)-5-carboxylicacid]); 6-Tetrachloro-Fluorescein([4,7,2′,7′-tetrachloro-(3′,6′-dipivaloylfluoresceinyl)-6-carboxylicacid]); 5-TAMRA (5-carboxytetramethylrhodamine; Xanthylium,9-(2,4-dicarboxyphenyl)-3,6-bis(dimethylamino); 6-TAMRA(6-carboxytetramethylrhodamine; Xanthylium, 9-(2,5-dicarboxyphenyl)-3,6-bis(dimethylamino); EDANS (5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid); 1,5-IAEDANS(5-((((2-iodoacetyl)amino)ethyl) amino)naphthalene-1-sulfonic acid);DABCYL (4-((4-(dimethylamino)phenyl) azo)benzoic acid) Cy5(Indodicarbocyanine-5) Cy3 (Indodicarbocyanine-3); and BODIPY FL(2,6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-proprionicacid), as well as suitable derivatives thereof.

In certain embodiments of the invention, a probe may also be labeledwith two chromophores, and a change in the absorption spectra of thelabel pair is used as a detection signal, as an alternative to measuringa change in fluorescence.

In the method of the invention, fluorescence intensity of the probe ismeasured at one or more wavelengths with a fluorescencespectrophotometer or microtitre plate reader, according to methods knownin the art.

C. Fluorescence Quenching Assay

A fluorescence quenching assay is useful in the invention for twopurposes. The first is to determine whether the secondary structure of aprobe is “stable” as defined herein. The second is to determine whetherthe secondary structure of the probe has undergone a “change” uponbinding of the probe to the target nucleic acid.

A probe according to the invention is labeled with a pair of interactivelabels (e.g., a FRET or non-FRET pair) wherein one member of the pair isa fluorophore and the other member of the pair is a quencher. Forexample, a probe according to the invention is labeled with afluorophore and a quencher and fluorescence is measured in the absenceof a target nucleic acid, over a range of temperatures, e.g., whereinthe lower temperature limit of the range is at least 50° Celsius below,and the upper temperature limit of the range is at least 50° Celsiusabove the Tm or the predicted Tm of the probe.

D. Stability

The “stability” of the secondary structure of a probe according to theinvention is determined as follows. A probe is labeled with a pair ofinteractive labels (either FRET or non-FRET pairs) described herein,according to methods well known in the art (for example, as described inGlazer and Mathies, 1997, Curr. Opin. Biotechnol., 8:94; Ju et al.,1995, Analytical Biochemistry, 231: 131)). The location of theinteractive labels on the probe is such that the labels are separatedwhen the secondary structure of the probe changes following binding ofthe probe to the target nucleic acid.

A standard curve for the probe (for example FIG. 13 e), whereinfluorescence is plotted versus temperature, is prepared by incubating asample comprising typically 125 nM probe in 1× Melting Buffer (20 mMTris-HCl, pH 8.0, 1 mM MgCl₂) or alternatively, in 5 mM Tris-HCl, pH8.0, 0.1 mM EDTA, or other appropriate buffers for a time that issufficient to permit denaturing and reannealing of the probe (typicallythe standard curve is generated using a fluorometer or spectrometer thatundergoes a 1° C. per minute change, and measuring the fluorescence in afluorometer or scanning fluorescence spectrophotometer over a range oftemperatures wherein the lower temperature limit of the range is atleast 50° C. below, and the upper temperature limit of the range is atleast 50° C. above the Tm or predicted Tm of the probe. The Tm of theprobe is predicted based on the base pair composition according tomethods well known in the art (see, Sambrook, supra; Ausubel, supra).

Standard curves are generated and compared, using a variety of buffers(e.g., 1×TNE buffer (10×-0.1M Tris base, 10 mM EDTA, 2.0 M NaCl, pH7.4), FEN nuclease buffer, described herein, 1× Cloned Pfu buffer,described herein, 1× Sentinel Molecular beacon buffer, described herein)including a buffer that is possible and preferentially optimal for theparticular nuclease to be employed in the cleavage reaction. The pH ofthe buffer will be monitored as the temperature increases, and adjustedas is needed.

The temperature of the fluorometer or spectrophotometer can becontrolled such that the fluorescence of the sample is measured atspecific temperatures. Fluorescence can be measured for example with aPerkin-Elmer LS50B Luminescence Spectrometer in combination with atemperature regulatable water bath (e.g., for example available fromFisher Scientific).

The stability of the secondary structure of a probe at a particulartemperature is determined by measuring the fluorescence of the probe ata particular temperature, as above, and determining if the value of thefluorescence is less than the fluorescence at the Tm, as determined fromthe standard curve. The secondary structure of the probe is “stable” ina FRET assay, at a temperature that is at or below the temperature ofthe cleavage reaction (i.e., at which cleavage is performed) if thelevel of fluorescence at the temperature at or below the temperature ofthe cleavage reaction is altered (i.e., at least 5%, preferably 20% andmost preferably 25% more or less than) the level of fluorescence at atemperature that is equal to the Tm of the probe. The secondarystructure of the probe is “stable” in a fluorescence quenching assay, ata temperature that is at or below the temperature of the cleavagereaction (i.e., at which cleavage is performed) if the level offluorescence at the temperature at or below the temperature of thecleavage reaction is altered (i.e., at least 5%, preferably 20% and mostpreferably 25% more or less than) the level of fluorescence at atemperature that is equal to the Tm of the probe. (see FIGS. 13 f and 9g).

Alternatively, the stability of the secondary structure of the probe isdetermined by modifying the method of Gelfand et al. (1999, Proc. Natl.Acad. Sci. USA, 96:6113), incorporated herein by reference, to determinethe fluorescence of a probe labeled with a pair of interactive labelsover a range of temperatures, as described hereinabove.

VII. Detecting a Secondary Structure

A secondary structure according to the invention is detected bygenerating a standard curve of fluorescence versus temperature for aprobe comprising a pair of interactive labels in a FRET assay, asdescribed above (see FIG. 13 e). A probe that exhibits a change influorescence that correlates with a change in temperature (see FIG. 13e) (e.g., fluorescence increases as the temperature of the FRET reactionis increased) is capable of forming a secondary structure.

VIII. Measuring a Change in Secondary Structure

A “change” in secondary structure according to the invention is detectedby analyzing a probe comprising a pair of interactive labels in a FRETor fluorescence quenching assay at a particular temperature below the Tmof the probe, (e.g., the cleaving temperature), as described above, inthe presence of absence of 100 nM to 10 μM of a target nucleic acidsequence (typically the target nucleic acid sequence is in a 2-4 molarexcess over the probe concentration, i.e., 250-500 nM target nucleicacid sequence is used).

Alternatively, a change in the secondary structure of the probe isdetermined by modifying the method of Gelfand et al. (1999, Proc. Natl.Acad. Sci. USA, 96:6113), incorporated herein by reference, to determinethe fluorescence of a probe labeled with a pair of interactive labels inthe presence or absence of a target nucleic acid as describedhereinabove.

A “change” in secondary structure that occurs when a probe according tothe invention binds to a target nucleic acid, is measured as an increasein fluorescence, such that the level of fluorescence after binding ofthe probe to the target nucleic acid at the temperature below the Tm ofthe probe, is greater than (e.g., at least 5%, preferably 5-20% and morepreferably 25 or more) the level of fluorescence observed in the absenceof a target nucleic acid sequence (see FIG. 13 g).

IX. Methods of Use

The invention provides for a method of detecting a target nucleic acidby linear amplification.

The invention also provides for a method of detecting a target nucleicacid by exponential amplification.

Both methods of the invention can be performed isothermally or performedunder conditions of thermal cycling, e.g., PCR, nucleic acidamplification.

X. Samples

The invention provides for a method of detecting or measuring a targetnucleic acid sequence in a sample, as defined herein. As used herein,“sample” refers to any substance containing or presumed to contain anucleic acid of interest (a target nucleic acid sequence) or which isitself a target nucleic acid sequence, containing or presumed to containa target nucleic acid sequence of interest. The term “sample” thusincludes a sample of target nucleic acid sequence (genomic DNA, cDNA orRNA), cell, organism, tissue, fluid or substance including but notlimited to, for example, plasma, serum, spinal fluid, lymph fluid,synovial fluid, urine, tears, stool, external secretions of the skin,respiratory, intestinal and genitourinary tracts, saliva, blood cells,tumors, organs, tissue, samples of in vitro cell culture constituents,natural isolates (such as drinking water, seawater, solid materials,)microbial specimens, and objects or specimens that have been “marked”with nucleic acid tracer molecules.

EXAMPLES

The invention is illustrated by the following nonlimiting exampleswherein the following materials and methods are employed. The entiredisclosure of each of the literature references cited hereinafter areincorporated by reference herein.

Example 1 Linear Isothermal Amplification

A target nucleic acid sequence can be detected and/or measured by thefollowing method of linear isothermal amplification.

Step 1:

A labeled first duplex is formed prior to the addition of a nuclease byincubating a sample containing a target nucleic acid (A′B′C′, FIG. 2), adownstream, 5′ radioactively, end labeled second oligonucleotide (FC,FIG. 2) comprising a 5′ region that is not complementary to the targetnucleic acid sequence, and an upstream first oligonucleotide (A, FIG. 2)under conditions that permit hybridization and formation of a firstduplex wherein the 5′ region of the second oligonucleotide is a flap andthe extension region of the target nucleic acid is not hybridized to theflap. For example, the sample is heated at 95° C. for 5 minutes and thencooled to approximately 50-60° C. Alternatively, the target nucleicacid, the second oligonucleotide and the first oligonucleotide arehybridized at a temperature (for example, 72° C. or 69° C.), that isoptimal for hybridization, and subsequent steps of polymerization andcleavage, and for a time sufficient to permit hybridization andformation of a first duplex, wherein the 5′ region of the probe is aflap and the extension region of the target nucleic acid is singlestranded, for example, 15 min-1 hour.

In certain embodiments of the invention, a first or second duplex isformed by incubating the components of a first or second duplex in thepresence of a denaturing agent (e.g., DMSO or glycerol) at aconcentration that is sufficient to permit hybridization and formationof a first or second duplex, according to the invention, as well assubsequent steps of polymerization and cleavage, described below. Aconcentration of a denaturing agent that is useful according to theinvention will vary depending on the base pair compositions of thecomponents of the duplex.

Step 2:

A labeled first cleavage structure is prepared as follows. A nucleicacid polymerization activity is added (for example Taq polymerase), andincubated under conditions that permit the polymerase to extend a firstoligonucleotide (A, FIG. 2) such that it is adjacent to the flap of thedownstream second oligonucleotide (FC, FIG. 2), for example 72° C. or69° C. in 1×Pfu buffer (Stratagene) for 5 minutes to 1 hour, therebyforming a first cleavage structure.

Step 3:

The labeled first cleavage structure prepared in Step 2 is cleaved witha preparation of PfuFEN-1 (i.e. cloned Pyrococcus furiosus FEN-1 that isprepared as described below in Example 5). Cleaving is performed at thesame temperature at which all polymerization and subsequenthybridization steps are performed, for example, 72° C. or 69° C.

For Example, cleavage is carried out by adding 2 μl of PfuFEN-1 to a 7μl reaction mixture containing the following:

3 μl cleavage structure (10 ng-10 μg) 0.7 μl 10x FEN nuclease buffer(10X FEN nuclease buffer contains 500 mM Tris-HCl pH 8.0, 100 mM MgCl₂)2.00 μl PfuFEN-1 enzyme or H₂O 1.3 μl H₂O 7.00 μl total volume

Samples are incubated for one hour at 72° C. or 69° C. in a Robocyler 96hot top thermal cycler.

Step 4:

A labeled second duplex is formed as follows. A sample containing atemplate nucleic acid (F′G′H′, FIG. 2) and an upstream, 5′radioactively, end labeled third oligonucleotide (FH, FIG. 2) comprisinga 5′ region that is not complementary to the template nucleic acidsequence, is incubated under conditions that permit hybridization of thetemplate nucleic acid and the third oligonucleotide (as described inStep 1). The hybridized template nucleic acid/third oligonucleotide iscombined with an amount of the sample containing the cleavage productsof the first cleavage structure, that is the released flap of the secondoligonucleotide (F, FIG. 2), prepared in Step 3, that is sufficient toform a second duplex wherein the 5′ region of the third oligonucleotideis a flap and the extension region of the template nucleic acid issingle stranded. The resulting mixture is incubated at 72° C. or 69° C.in the presence or absence of added denaturing agent (as above), for atime sufficient to permit hybridization, for example, 15 min-1 hour.

Step 5:

A labeled second cleavage structure is prepared as follows. A nucleicacid polymerization activity is added (for example Taq polymerase), andincubated under conditions that permit the polymerase to extend the flapof the second oligonucleotide (F, FIG. 2) such that it is adjacent tothe flap of the downstream third oligonucleotide, for example 72° C. or69° C. in 1×Pfu buffer (Stratagene) for 5 minutes to 1 hour, therebyforming a second cleavage structure.

Step 6:

The labeled second cleavage structure prepared in Step 5 is cleaved witha preparation of PfuFEN-1, as described in step 3.

Step 7:

Following the addition of 2 μl of Sequencing Stop dye solution (includedin the Stratagene Cyclist DNA sequencing kit, catalog #200326), samplesare heated at 99° C. for five minutes. Released, labeled, flaps areanalyzed by gel electrophoresis as follows. Samples are loaded on aneleven inch long, hand-poured, 20% acrylamide/bis acrylamide, 7M ureagel. The gel is run at 20 watts until the bromophenol blue has migratedapproximately ⅔ the total distance. The gel is removed from the glassplates and soaked for 10 minutes in fix solution (15% methanol, 5%acetic acid) and then for 10 minutes in water. The gel is placed onWhatmann 3 mm paper, covered with plastic wrap and dried for 2 hours ina heated vacuum gel dryer (˜800 C). The gel is exposed overnight toX-ray film to detect the presence of a signal that is indicative of thepresence of a target nucleic acid sequence.

Alternatively, extension and cleavage are performed with an enzyme thatexhibits both activities. The polymerization activity employed in step 2can be different from the polymerization activity employed in step 5.The cleavage means employed in step 3 can be different from the cleavagemeans of step 6.

Example 2 Linear Isothermal Amplification

A target nucleic acid sequence can be detected and/or measured by thefollowing method of linear isothermal amplification.

Step 1:

A labeled first duplex is formed prior to the addition of a nuclease byincubating a sample containing a target nucleic acid (A′B′C′, FIG. 2), adownstream, 5′ radioactively, end labeled second oligonucleotide (FC,FIG. 2) comprising a 5′ region that is complementary to the targetnucleic acid sequence, and an upstream first oligonucleotide (A, FIG. 2)under conditions that permit hybridization and formation of a firstduplex wherein the 5′ region of the second oligonucleotide is hybridizedto the target nucleic acid. For example, the sample is heated at 95° C.for 5 minutes and then cooled to approximately 50-60° C. The first andsecond oligonucleotide hybridize to non-overlapping regions of thetarget nucleic acid. Alternatively, the target nucleic acid, the secondoligonucleotide and the first oligonucleotide are hybridized at atemperature (for example, 72° C. or 69° C. that is optimal forhybridization, and subsequent steps of polymerization and cleavage, andfor a time sufficient to permit hybridization and formation of a firstduplex, wherein the 5′ region of the probe is hybridized to the targetnucleic acid, for example, 15 min-1 hour.

In certain embodiments of the invention, a first or second duplex isformed by incubating the components of a first or second duplex in thepresence of a denaturing agent (e.g., DMSO or glycerol) as described instep 1 of Example 1.

Step 2:

A labeled first cleavage structure is prepared as follows. A nucleicacid polymerization activity with strand displacement activity is added(for example Vent DNA polymerase), and incubated under conditions thatpermit the polymerase to extend a first oligonucleotide (A, FIG. 2) suchthat it partially displaces the 5′ end of the second oligonucleotide FC(for example 72° C. or 69° C. in 1× ThermoPol Buffer (10 mM KCL, 20 mMTris-HCl (pH 8.8 at 25° C.), 10 mM (NH₄)₂SO₄, 2 mM MgSO₄, 0.1% TritonX-100, New England Biolabs) for 5 minutes to 1 hour), thereby forming afirst cleavage structure. The displaced region of oligonucleotide FC,that is F, forms a 5′ flap that is cleaved upon the addition of anuclease (e.g., a FEN nuclease).

Step 3:

The labeled first cleavage structure prepared in Step 2 is cleaved witha preparation of PfuFEN-1 (i.e. cloned Pyrococcus furiosus FEN-1 that isprepared as described below in Example 5). Cleaving is performed at thesame temperature at which all polymerization and subsequenthybridization steps are performed, for example, 72° C. or 69° C.

For Example, cleavage is carried out by adding 2 μl of PfuFEN-1 to a 7μl reaction mixture containing the following:

3 μl cleavage structure (10 ng-10 μg) 0.7 μl 10x FEN nuclease buffer(10X FEN nuclease buffer contains 500 mM Tris-HCl pH 8.0, 100 mM MgCl₂)2.00 μl PfuFEN-1 enzyme or H₂O 1.3 μl H₂O 7.00 μl total volume

Samples are incubated for one hour at 72° C. or 69° C. in a Robocyler 96hot top thermal cycler.

Step 4:

A labeled second duplex is formed as follows. A sample containing atemplate nucleic acid (F′G′H′, FIG. 2) and an upstream, 5′radioactively, end labeled third oligonucleotide (FH, FIG. 2) comprisinga 5′ region that is complementary to the template nucleic acid sequenceis incubated under conditions that permit hybridization of the templatenucleic acid and the third oligonucleotide (as described in Step 1). Thehybridized template nucleic acid/third oligonucleotide is combined withan amount of the sample containing the cleavage products of the firstcleavage structure, that is the released flap of the secondoligonucleotide (F, FIG. 2), prepared in Step 3, that is sufficient toform a second duplex wherein the 5′ region of the third oligonucleotideis hybridized to the template nucleic acid. The resulting mixture isincubated at 72° C. or 69° C. in the presence or absence of addeddenaturing agent (as above), for a time sufficient to permithybridization, for example, 15 min-1 hour. The flap of the secondoligonucleotide and the third oligonucleotide hybridize tonon-overlapping regions of the template nucleic acid.

Step 5:

A labeled second cleavage structure is prepared as follows. A nucleicacid polymerization activity is added (for example VENT DNA polymerase),and incubated under conditions that permit the polymerase to extend theflap of the second oligonucleotide (F, FIG. 2) such that it partiallydisplaces the 5′ end of the third oligonucleotide FH (as in step 2,above), thereby forming a second cleavage structure. The displacedregion of oligonucleotide FH, that is F, forms a 5′ flap that is cleavedupon the addition of a FEN nuclease.

Step 6:

The labeled second cleavage structure prepared in Step 5 is cleaved witha preparation of PfuFEN-1, as described in step 3.

Step 7:

Following the addition of 2 μl of Sequencing Stop dye solution (includedin the Stratagene Cyclist DNA sequencing kit, catalog #200326), samplesare heated at 99° C. for five minutes. Released, labeled, flaps areanalyzed by gel electrophoresis as follows. Samples are loaded on aneleven inch long, hand-poured, 20% acrylamide/bis acrylamide, 7M ureagel. The gel is run at 20 watts until the bromophenol blue has migratedapproximately ⅔ the total distance. The gel is removed from the glassplates and soaked for 10 minutes in fix solution (15% methanol, 5%acetic acid) and then for 10 minutes in water. The gel is placed onWhatmann 3 mm paper, covered with plastic wrap and dried for 2 hours ina heated vacuum gel dryer (˜800 C). The gel is exposed overnight toX-ray film to detect the presence of a signal that is indicative of thepresence of a target nucleic acid sequence.

Alternatively, extension and cleavage are performed with an enzyme thatexhibits both activities. The polymerization activity employed in step 2can be different from the polymerization activity employed in step 5.The cleavage means employed in step 3 can be different from the cleavagemeans of step 6.

Example 3 Exponential Isothermal Amplification

A target nucleic acid sequence can be detected and/or measured by thefollowing method of exponential isothermal amplification.

Steps 1-3 of Example 1 are performed.

Step 4:

A labeled second duplex is formed as follows. A sample containing atemplate nucleic acid (F′G1′H1′G2′H2′, FIG. 5), an upstream, 5′radioactively, end labeled third oligonucleotide (FH1, FIG. 5)comprising a 5′ region that is not complementary to the template nucleicacid sequence, and an upstream, 5′ radioactively, end labeled fourtholigonucleotide (FH2, FIG. 5) comprising a 5′ region that is notcomplementary to the template nucleic acid sequence, is incubated underconditions that permit hybridization of the template nucleic acid andthe third and fourth oligonucleotides (as described in Step 1 of Example1). The hybridized template nucleic acid/third and fourtholigonucleotides is combined with an amount of the sample containing thecleavage products of the first cleavage structure, that is the releasedflap of the second oligonucleotide (F, FIG. 5), prepared in Step 3, thatis sufficient to form a second duplex wherein the 5′ regions of thethird and fourth oligonucleotides are flaps and the extension regions ofthe template nucleic acid are single stranded. The resulting mixture isincubated at 72° C. or 69° C. in the presence or absence of addeddenaturing agent (as above), for a time sufficient to permithybridization, for example, 15 min-1 hour.

Step 5:

A labeled second cleavage structure is prepared as follows. A nucleicacid polymerization activity is added (for example Taq polymerase), andincubated under conditions that permit the polymerase to extend the flapof the second oligonucleotide (F, FIG. 5) such that it is adjacent tothe flap of the downstream third oligonucleotide (FH1, FIG. 5), forexample 72° C. or 69° C. in 1×Pfu buffer (Stratagene) for 5 minutes to 1hour, thereby forming a second cleavage structure.

Step 6:

The labeled second cleavage structure prepared in Step 5 is cleaved witha preparation of PfuFEN-1, as described in step 3 of Example 1 torelease the flap of the third oligonucleotide (F, FIG. 5, step 2).

Step 7:

A labeled third cleavage structure is prepared as follows. A nucleicacid polymerization activity is added (for example Taq polymerase), andincubated under conditions that permit the polymerase to extend the flapof the second oligonucleotide (F, FIG. 5) such that it is adjacent tothe flap of the downstream fourth oligonucleotide, for example 72° C. or69° C. in 1×Pfu buffer (Stratagene) for 5 minutes to 1 hour, therebyforming a third cleavage structure.

Step 8:

The labeled second cleavage structure prepared in Step 5 is cleaved witha preparation of PfuFEN-1, as described in step 3 of Example 1 torelease the flap of the fourth oligonucleotide (F, FIG. 5, step 4).

Step 9:

Following the addition of 2 μl of Sequencing Stop dye solution (includedin the Stratagene Cyclist DNA sequencing kit, catalog #200326), samplesare heated at 99° C. for five minutes. Released, labeled, flaps areanalyzed by gel electrophoresis as follows. Samples are loaded on aneleven inch long, hand-poured, 20% acrylamide/bis acrylamide, 7M ureagel. The gel is run at 20 watts until the bromophenol blue has migratedapproximately ⅔ the total distance. The gel is removed from the glassplates and soaked for 10 minutes in fix solution (15% methanol, 5%acetic acid) and then for 10 minutes in water. The gel is placed onWhatmann 3 mm paper, covered with plastic wrap and dried for 2 hours ina heated vacuum gel dryer (˜800 C). The gel is exposed overnight toX-ray film to detect the presence of a signal that is indicative of thepresence of a target nucleic acid sequence.

Alternatively, extension and cleavage are performed with an enzyme thatexhibits both activities. The polymerization activity employed in step 2can be different from the polymerization activity employed in step 5 andthe polymerization activity of step 7. The cleavage means employed instep 3 can be different from the cleavage means of step 6 and thecleavage means of step 8.

Example 4 Exponential Isothermal Amplification

A target nucleic acid sequence can be detected and/or measured by thefollowing method of exponential isothermal amplification.

Steps 1-3 of Example 2 are performed.

Step 4:

A labeled second duplex is formed as follows. A sample containing atemplate nucleic acid (F′G1′H1′G2′H2′, FIG. 5), an upstream, 5′radioactively, end labeled third oligonucleotide (FH1, FIG. 5)comprising a 5′ region that is at least partially complementary to thetemplate nucleic acid sequence, and an upstream, 5′ radioactively, endlabeled fourth oligonucleotide (FH2, FIG. 5) comprising a 5′ region thatis complementary to the template nucleic acid sequence, is incubatedunder conditions that permit hybridization of the template nucleic acidand the third and fourth oligonucleotides (as described in Step 1 ofExample 1). The hybridized template nucleic acid/third and fourtholigonucleotides is combined with an amount of the sample containing thecleavage products of the first cleavage structure, that is the releasedflap of the second oligonucleotide (F, FIG. 5), prepared in Step 3, thatis sufficient to form a second duplex wherein the 5′ regions of thethird and fourth oligonucleotides are hybridized to the template nucleicacid. The resulting mixture is incubated at 72° C. or 69° C. in thepresence or absence of added denaturing agent (as above), for a timesufficient to permit hybridization, for example, 15 min-1 hour. Each ofthe flap of the second oligonucleotide, the third oligonucleotide andthe fourth oligonucleotide hybridize to non-overlapping regions of thetemplate nucleic acid.

Step 5:

A labeled second cleavage structure is prepared as follows. A nucleicacid polymerization activity is added (for example VENT DNA polymerase),and incubated under conditions that permit the polymerase to extend theflap of the second oligonucleotide (F, FIG. 5) such that it partiallydisplaces the 5′ end of the third oligonucleotide FH1 (as in step 2,above), thereby forming a second cleavage structure. The displacedregion of oligonucleotide FH1, that is F, forms a 5′ flap that iscleaved upon the addition of a nuclease (e.g., a FEN nuclease).

Step 6:

The labeled second cleavage structure prepared in Step 5 is cleaved witha preparation of PfuFEN-1, as described in step 3 of Example 2 torelease the flap of the third oligonucleotide (F, FIG. 5, step 2).

Step 7:

A labeled third cleavage structure is prepared as follows. A nucleicacid polymerization activity is added (for example VENT DNA polymerase),and incubated under conditions that permit the polymerase to extend theflap of the second oligonucleotide (F, FIG. 5) such that it partiallydisplaces the 5′ end of the fourth oligonucleotide FH2 (as in step 2,above), thereby forming a third cleavage structure. The displaced regionof oligonucleotide FH2, that is F, forms a 5′ flap that is cleaved uponthe addition of a nuclease (e.g., a FEN nuclease).

Step 8:

The labeled third cleavage structure prepared in Step 7 is cleaved witha preparation of PfuFEN-1, as described in step 3 of Example 2 torelease the flap of the fourth oligonucleotide (F, FIG. 5, step 4).

Step 9:

Following the addition of 2 μl of Sequencing Stop dye solution (includedin the Stratagene Cyclist DNA sequencing kit, catalog #200326), samplesare heated at 99° C. for five minutes. Released, labeled, flaps areanalyzed by gel electrophoresis as follows. Samples are loaded on aneleven inch long, hand-poured, 20% acrylamide/bis acrylamide, 7M ureagel. The gel is run at 20 watts until the bromophenol blue has migratedapproximately ⅔ the total distance. The gel is removed from the glassplates and soaked for 10 minutes in fix solution (15% methanol, 5%acetic acid) and then for 10 minutes in water. The gel is placed onWhatmann 3 mm paper, covered with plastic wrap and dried for 2 hours ina heated vacuum gel dryer (˜800 C). The gel is exposed overnight toX-ray film to detect the presence of a signal that is indicative of thepresence of a target nucleic acid sequence.

Alternatively, extension and cleavage are performed with an enzyme thatexhibits both activities. The polymerization activity employed in step 2can be different from the polymerization activity employed in step 5 andthe polymerization activity of step 7. The cleavage means employed instep 3 can be different from the cleavage means of step 6 and thecleavage means of step 8.

Example 5 Cloning Pfu FEN-1

A thermostable FEN nuclease enzyme useful according to the invention canbe prepared according to the following method.

The thermostable FEN nuclease gene can be isolated from genomic DNAderived from P. furiosus (ATCC#43587) according to methods of PCRcloning well known in the art. The cloned PfuFEN-1 can be overexpressedin bacterial cells according to methods well known in the art anddescribed below.

The following pCAL-n-EK cloning oligonucleotides were synthesized andpurified:

a. 5′GACGACGACAAGATGGGTGTCCCAATTGGTGAGATTATACCAAGAAAA G 3′ and b.5′GGAACAAGACCCGTTTATCTCTTGAACCAACTTTCAAGGGTTGATTGT TTTCCACT 3′.

The Affinity® Protein Expression and Purification System was obtainedfrom Stratagene and used according to the manufacturer's protocols.

Amplification

The insert DNA was prepared by PCR amplification with gene-specificprimers (oligonucleotides a and b, described above) that include 12 and13-nucleotide sequences at the 5′ ends that are complementary to thepCAL-n-EK vector single-stranded tails, thus allowing for directionalcloning. The FEN-1 sequence was amplified from genomic DNA derived fromP. furiosus by preparing amplification reactions (five independent 100μl reactions) containing:

50 μl 10x cPfu Buffer (Stratagene) 7.5 μl Pfu Genomic DNA (approx. 100ng/μl) 7.5 μl PfuTurbo(2.5 u/μl), (Stratagene, Catalog # 600250) 15 μlmixed primer pair (100 ng/μl each) (oligonucleotides a and b, describedabove) 4 μl 100 mM dNTP 416 μl H₂O 500 μl totaland carrying out the amplification under the following conditions usinga Stratagene Robocycler 96 hot top thermal cycler:

Window 1 95° C. 1 minute 1 cycle Window 2 95° C. 1 minute 50° C. 1minute 30 cycles 72° C. 3 minutes

The PCR products from each of the five reactions were combined into onetube, purified using StrataPrep PCR and eluted in 50 μl 1 mM Tris-HCl pH8.6. The FEN-1 PCR product was analyzed on a gel and was determined tobe approximately 1000 bp.

The PCR product comprising the fen-1 gene was cloned into the pCALnEKLIC vector (Stratagene) by creating ligation independent cloning termini(LIC), annealing the PCR product comprising the fen-1 gene to thepCALnEK LIC vector (Stratagene), and transforming cells with theannealing mixture according to the following method. Briefly, followingPCR amplification, the PCR product is purified and treated with Pfu DNApolymerase in the presence of dATP (according to the manual includedwith the Affinity® Protein Expression and Purification System,Stratagene, catalog #200326). In the absence of dTTP, dGTP and dCTP, the3′ to 5′-exonuclease activity of Pfu DNA polymerase removes at least 12and 13 nucleotides at the respective 3′ ends of the PCR product. Thisactivity continues until the first adenine is encountered, producing aDNA fragment with 5′-extended single-stranded tails that arecomplementary to the single-stranded tails of the pCAL-n-EK vector.

Creating LIC Termini

LIC termini were created by preparing the following mixture:

45 μl purified PCR product (˜0.5 μg/μl)

2.5 μl 10 mM dATP

5 μl 10× cPfu buffer

1 μl cPfu (2.5 u/μl)

0.5 μl H₂O

cPfu and cPfu buffer can be obtained from Stratagene (cPfu, StratageneCatalog #600153 and cPfu buffer, Stratagene Catalog #200532).

Samples were incubated at 72° C. for 20 minutes and products were cooledto room temperature. To each sample was added 40 ng prepared pCALnEK LICvector (the prepared vector is available commercially from Stratagene inthe Affinity LIC Cloning and Protein Purification Kit (214405)). Thevector and insert DNA are combined, allowed to anneal at roomtemperature and transformed into highly competent bacterial host cells(Wyborski et al., 1997, Strategies, 10:1).

Preparing Cells for Production of FEN

Two liters of LB-AMP was inoculated with 20 ml of an overnight cultureof a FEN-1 clone (clone 3). Growth was allowed to proceed forapproximately 11 hours at which point cells had reached an OD₆₀₀=0.974.Cells were induced overnight (about 12 hours) with 1 mM IPTG. Cells werecollected by centrifugation and the resulting cell paste was stored at−20° C.

Purification of tagged FEN-1

Cells were resuspended in 20 ml of Calcium binding buffer

CaCl₂ binding Buffer

50 mM Tris-HCl (pH 8.0)

150 mM NaCl

1.0 mM MgOAc

2 mM CaCl₂

The samples were sonicated with a Branson Sonicator using a microtip.The output setting was 5 and the duty cycle was 90%. Samples weresonicated three times and allowed to rest on ice during the intervals.The sonicate was centrifuged at 26,890×g. Cleared supernatants weremixed with 1 ml of washed (in CaCl₂ binding buffer) calmodulin agarose(CAM agarose) in a 50 ml conical tube and incubated on a slowly rotatingwheel in a cold room (40 C) for 5 hours. The CAM agarose was collectedby light centrifugation (5000 rpm in a table top centrifuge).

Following removal of the supernatant, the CAM agarose was washed with 50ml CaCl₂ binding buffer and transferred to a disposable drip column. Theoriginal container and pipet were rinsed thoroughly to remove residualagarose. The column was rinsed with approximately 200 ml of CaCl₂binding buffer.

Elution was carried out with 10 ml of 50 mM NaCl elution buffer (50 mMNaCl, 50 mM Tris-HCl pH 8.0, 2 mM EGTA). 0.5 ml fractions werecollected. A second elution step was carried out with 1M NaCl elutionbuffer wherein 0.5 ml fractions were collected.

Evaluation of Purified Tagged FEN-1

Fractions containing CBP-tagged Pfu FEN-1 eluted in 1M NaCl were boiledin SDS and analyzed by SDS-PAGE on a 4-20% gel stained with Sypro Orange(FIG. 15).

The protein concentration of uncleaved FEN-1 was determined to beapproximately 150 ng/microliter (below).

Enterokinase Protease (EK) Cleavage of the Purified FEN-1

Fractions 3-9 were dialyzed in 50 mM NaCl, 50 mM Tris-HCl pH 8.0 and 2mM CaCl₂ overnight at 4° C.

An opaque, very fine precipitate appeared in the dialyzed FEN-1. Whenthe sample was diluted 1/20 the precipitate was removed. When the samplewas diluted ⅓ insoluble material was still detectable. The ⅓ dilutedmaterial was heated at 37° C. for 2 minutes and mixed with Tween 20 to afinal concentration of 0.1%. Upon the addition of the Tween 20, therewas an almost immediate formation of “strings” and much coarser solidsin the solution which could not be reversed even after the solution wasadjusted to 1M NaCl.

EK cleavage was carried out using as a substrate the sample that wasdiluted 1/20 as well as with a dilute sample prepared by rinsing thedialysis bag with 1×EK buffer. EK cleavage was carried out by theaddition of 1 μl EK (1 u/μl) overnight at room temperature (about 16hours).

100 μl of STI agarose combined with 100 μl of CAM agarose were rinsedtwice with 10 ml of 1×STI buffer (50 mM Tris-HCl pH 8.0, 200 mM NaCl, 2mM CaCl₂, 0.1% Tween 20). NaCl was added to the two EK samples to bringthe final concentration to 200 mM NaCl. The two samples were combinedand added to the rinsed agarose. The samples were rotated slowly on awheel at 4° C. for three hours and separated by light centrifugation ina table top centrifuge (as described). The supernatant was removed andthe resin was rinsed twice with 500 μl 1×STI. The two rinses werecombined and saved separately from the original supernatant. Sampleswere analyzed by SDS-PAGE on a 4-20% gel.

The concentration of digested product was approximately 23 ng/μl asdetermined by comparison to a Pfu standard at a concentration ofapproximately 50 ng/ml.

Example 6 FEN Nuclease Activity

The endonuclease activity of a FEN nuclease and the cleavage structurerequirements of a FEN nuclease prepared as described in Example 5 can bedetermined according to the methods described either in the sectionentitled “FEN nucleases” or below.

Briefly, three templates (FIG. 14) are used to evaluate the activity ofa FEN nuclease according to the invention. Template 1 is a 5′ ³³Plabeled oligonucleotide (Heltest4) with the following sequence:

5′ AAAATAAATAAAAAAAATACTGTTGGGAAGGGCGATCGGTGCG3′.

The underlined section of Heltest4 represents the region complementaryto M13mp 18+. The cleavage product is an 18 nucleotide fragment with thesequence AAAATAAATAAAAAAAAT. Heltest4 binds to M13 to produce acomplementary double stranded domain as well as a non-complementary 5′overhang. This duplex forms template 2 (FIG. 14). Template 3 (FIG. 14)has an additional primer (FENAS) bound to M13 which is directly adjacentto Heltest 4. The sequence of FENAS is: 5′CCATTCGCCATTCAGGCTGCGCA 3′. Inthe presence of template 3, a FEN nuclease binds the free 5′ terminus ofHeltest4, migrates to the junction and cleaves Heltest4 to produce an 18nucleotide fragment. The resulting cleavage products are separated on a6% acrylamide, 7M urea sequencing gel.

Templates are prepared as described below:

Template 1 Template 2 Template 3 Heltest4 14 μl 14 μl 14 μl M13 ** 14 μl14 μl FENAS ** ** 14 μl H₂O 28 μl 14 μl ** 10x Pfu Buff. 4.6 μl  4.6 μl 4.6 μl  Pfu buffer can be obtained from Stratagene (Catalog #200536).

The template mixture is heated at 95° C. for five minutes, cooled toroom temperature for 45 minutes and stored at 4° C. overnight.

The enzyme samples are as follows:

-   A. H₂O (control)-   B. 2 μl undiluted uncleaved FEN-1 (˜445 ng/μl)-   C. 2 μl 1/10 dilution of uncleaved FEN-1 (˜44.5 ng/μl)-   D. 2 μl enterokinase protease (EK) cleaved FEN-1 (˜23 ng/μl)

The four reaction mixtures are mixed with the three templates asfollows:

3 μl template 1, template 2 or template 3 0.7 μl 10x cloned Pfu buffer0.6 μl 100 mM MgCl₂ 2.00 μl FEN-1 or H₂O 0.7 μl H₂O 7.00 μl total volume

The reactions are allowed to proceed for 30 minutes at 50° C. andstopped by the addition of 2 μl formamide “Sequencing Stop” solution toeach sample. Samples are heated at 95° C. for five minutes and loaded ona 6% acrylamide 7M urea CastAway gel (Stratagene).

Alternatively, FEN nuclease activity can be analyzed in the followingbuffer wherein a one hour incubation time is utilized.

10×FEN Nuclease Buffer

500 mM Tris-HCl pH 8.0

100 mM MgCl₂

The reaction mixture is as follows:

3 μl template 1, template 2 or template 3 0.7 μl 10x FEN nuclease buffer2.00 μl FEN-1 or H₂O (A-D, above) 1.3 μl H₂O 7.00 μl total volume

Samples are incubated for one hour at 50° C. in the Robocyler 96 hot topthermal cycler. Following the addition of 2 μl of Sequencing Stop (95%formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol,available from Stratagene) dye solution, samples are heated at 99° C.for five minutes. Samples are loaded on an eleven inch long,hand-poured, 20% acrylamide/bis acrylamide, 7M urea gel. The gel is runat 20 watts until the bromophenol blue has migrated approximately ⅔ thetotal distance. The gel is removed from the glass plates and soaked for10 minutes in fix solution (15% methanol, 5% acetic acid) and then for10 minutes in water. The gel is placed on Whatmann 3 mm paper, coveredwith plastic wrap and dried for 2 hours in a heated vacuum gel dryer(˜800 C). The gel is exposed overnight to X-ray film.

An autoradiograph of a FEN-1 nuclease assay wherein templates 1, 2 and 3(prepared as described above) are cleaved by the addition of:

-   A. H₂O-   B. 2 μl of CBP-tagged Pfu FEN-1-   C. 2 μl of CBP-tagged Pfu FEN-1 diluted (1:10)-   D. 2 μl of EK cleaved Pfu FEN-1    is presented in FIG. 16.

The lanes are as follows. Lanes 1A, 1B, 1C and 1D represent template 1cleaved with H₂O, undiluted CBP-tagged Pfu FEN-1, a 1:10 dilution ofCBP-tagged Pfu FEN-1 and EK cleaved Pfu FEN-1, respectively. Lanes 2A,2B, 2C and 2D represent template 2 cleaved with H₂O, undilutedCBP-tagged Pfu FEN-1, a 1:10 dilution of CBP-tagged Pfu FEN-1 and EKcleaved Pfu FEN-1, respectively. Lanes 3A, 3B, 3C and 3D representtemplate 3 cleaved with H₂O, undiluted CBP-tagged Pfu FEN-1, a 1:10dilution of CBP-tagged Pfu FEN-1 and EK cleaved Pfu FEN-1, respectively.

Tagged Pfu FEN-1 contains the N-terminal CBP affinity purification tag.Any differences in activity between tagged and untagged versions ofFEN-1 are due to differences in protein concentration (concentrations ofenzyme samples are provided above) since the amounts of tagged versusuntagged FEN-1 are not equivalent. Both tagged and untagged Pfu FEN-1demonstrate cleavage activity.

FIG. 16 demonstrates the background level of cleavage in the absence ofFEN-1 (lanes 1A, 2A and 3A). Further, this figure demonstrates thattagged Pfu FEN-1 cleaves more of template 2 as compared to template 1.In particular, the greatest amount of template 2 is cleaved in thepresence of undiluted, tagged Pfu FEN-1 (lane 2B). Analysis of template3 demonstrates that the greatest amount of template 3 is cleaved byundiluted, tagged Pfu FEN-1 and the least amount of template 3 iscleaved by diluted tagged FEN-1. Labeled probe migrates as a 40-43nucleotide band. FEN-1 preferentially cleaves template 3 (whichcomprises an upstream primer) as compared to template 2. The cleavageproduct bands are the major bands migrating at 16-20 nucleotides.Heterogeneity in the labeled cleavage products is the result ofheterogeneity in the labeled substrate, which was not gel-purified priorto use.

Example 7 Target Nucleic Acid Detection Using Nucleic Acid AmplificationReaction Conditions

A target nuclei acid sequence can be detected and/or measured by thefollowing method.

Step 1: Prepare reaction mixture

One or more reactions are prepared that contain the oligonucleotides ofthe invention. The oligonucleotides include a first oligonucleotide (A,FIG. 17), a second oligonucleotide (F-C, FIG. 17), a target nucleic acid(A′B′C′, FIG. 17), a template nucleic acid (F′G′H′, FIG. 17), and athird oligonucleotide (H, FIG. 17). The second oligonucleotide andtemplate nucleic acid are blocked at the 3′ nucleotide. The thirdoligonucleotide and the template nucleic acid are each coupled to onemember of a pair of interactive labels. In the embodiment depicted inFIG. 17, the third oligonucleotide is coupled to a fluorescer on its 3′end and the template nucleic acid is coupled to a quencher on its 5′end. The oligonucleotides are added to a reaction mixture containingstandard PCR reagents as well as a cleavage enzyme. The amplificationand detection reaction can be performed with the FullVelocity™ QPCRMaster Mix (Stratagene, Catalog No. 600561). This system is describedfurther in U.S. Pat. Nos. 6,528,254 and 6,548,250 (each incorporatedherein by reference in their entirety). Typical reaction conditions towhich the oligonucleotides are added include:

1X FullVelocity buffer (containing dNTPs) 5 U Pfu V93R exo(−)polymerase, 200 ng FEN-1 endonuclease

The reaction mixture is placed in a thermal cycler. Thermal cyclers areknown in the art and include the Mx3000p real-time PCR instrument(Stratagene. Calif.). Typical cycling parameters include: an initial 2min at 95° C. for 1 cycle, followed by 50 cycles of 10 sec at 95° C., 30sec at 60° C.

Step 2:

The oligonucleotides are denatured at 95° C. for either 2 minutes or 10sec.

Step 3:

A first duplex is formed by incubating the reaction mixture underconditions that permit hybridization and formation of a first duplexwherein the 5′ region of the second oligonucleotide is a flap and theextension region of the target nucleic acid is not hybridized to theflap. For example, the duplex is formed when the reaction mixture iscooled to approximately 50-60° C. for a time sufficient to permithybridization and formation of the first duplex. The first duplexincludes the target nucleic acid, the first oligonucleotide and thesecond oligonucleotide. See FIG. 17 A. The first oligonucleotidehybridizes to the first region of the target nucleic acid (A′; FIG.17A). The 3′ region of the second oligonucleotide hybridizes to thesecond region of the target nucleic acid (C′; FIG. 17A), while the 5′region of the second oligonucleotide forms a flap (F; FIG. 17A). Theextension region of the target nucleic acid is single stranded (B′; FIG.17A). The flap is complementary to a first region of the templatenucleic acid (F′; FIG. 17D).

Step 4:

A first cleavage structure is prepared as follows. The reaction isincubated under conditions that permit the polymerase to extend thefirst oligonucleotide such that it is adjacent to the flap of thedownstream second oligonucleotide. See FIG. 17B. The extension may beperformed at the same temperature as that applied during the formationof the first duplex or at a distinct temperature which allowspolymerization. The temperature is applied for a time sufficient topermit extension of the first oligonucleotide into the extension regionof the target nucleic acid (B′, FIG. 17B). The polymerase displaces thesecond oligonucleotide

Step 5:

The first cleavage structure is cleaved with the PfuFEN-1 of theFullVelocity multi-enzyme formulation. See FIG. 17C. Preferably thecleavage reaction is performed at the same temperature at which allpolymerization and/or hybridization steps are performed, e.g., 60 C. Thecleavage reaction releases the 5′ flap of the second oligonucleotide (F,FIG. 17C).

Step 6:

A second duplex is formed as follows. The second duplex is formed byincubating the reaction mixture under conditions that permithybridization and formation of a second duplex. The conditions arepreferably the same as those that result in the formation of the firstduplex (Step 3). The second duplex may form simultaneously with thefirst duplex. The second duplex is formed when the cleaved flap from thesecond oligonucleotide (F′; FIG. 17D) hybridizes to the first region ofthe template nucleic acid (F′; FIG. 17D) and the third oligonucleotide(H; FIG. 17D) hybridizes to the second region (H′; FIG. 17D) of thetemplate nucleic acid. Formation of the second duplex allows thereporter and quencher moieties operably coupled to the template nucleicacid and the third oligonucleotide to interact; substantially quenchingthe reporter.

Step 7:

A labeled second cleavage structure is prepared as follows. The reactionis incubated under conditions that permit the polymerase to extend thereleased flap of the second oligonucleotide (A, FIG. 17E). Theseconditions are preferably the same as described in Step 4 for the firstcleavage structure. The flap is extended, by the polymerase activity,into the extension region of the template nucleic acid. The polymerasepartially displaces the third oligonucleotide. (FIG. 17E).

Step 8:

The second cleavage structure is cleaved in a similar manner to thatdescribed in step 5 for the first cleavage reaction. However, duringthis cleavage reaction a detectable signal is produced, e.g.,fluorescence. See FIG. 17F. The signal is produced when the thirdoligonucleotide is cleaved causing the third oligonucleotide andtemplate nucleic acid to dissociate. Separation of the pair ofinteractive labels results in a detectable signal.

The reaction may be repeated for one or more additional cycles.

Example 8 Real-Time QPCR Detection Utilizing the Oligonucleotides of theInvention

The following experiment was performed to detect a target nucleic acid.

Primers:

HPRT-Fwd: 5′-AATTATGGACAGGACTGAACGTCTTGCT-3′ HPRT-Rev:5′-TCCAGCAGGTCAGCAAAGAATTTATAGC-3′

Probes:

Second Oligonucleotide:

5′--BHQ2- CAGTTCAGGGGCCTCATCTCGGTTTTTCCCAGATGAGCCCCCAGAACTC -FAM-3′

The underlined portion of the second oligonucleotide corresponds to the5′ flap that that was released in the cleavage reaction. The flap wascomplementary to the first portion of the template nucleic acid andserved as a primer in subsequent extension reactions.

Third Oligonucleotide:

5′-tGaAcGaTgcc-Hex-3′

The third oligonucleotide is complementary to the second portion of thetemplate nucleic acid.

Template Nucleic Acid:

5′--BHQ2- GGCATCGTTCA AAAGGAAAAACCGAGATGAGGCC-PO3-3′

The template nucleic acid had a first region (bold), which wascomplementary to and hybridizes with the released flap, an extensionregion and a second region, which was complementary to and hybridizedwith the third oligonucleotide (underlined). The template nucleic acidwas blocked at the 3′ nucleotide and was coupled to a quencher at the 5′nucleotide.

The oligonucleotides described above were used in a FullVelocity Q-PCRreaction (Stratagene) according to the manufacturer's instructions.Briefly, the following reagents were combined in the reaction mixture:1×FullVelocity buffer (containing dNTPs), Xul of the FullVelocityMulti-enzyme formulation, 400 nM the HPRT Fwd primer and 200 nM of theHPRT Rev primer, 400 nM of the second oligonucleotide, 300 nM of thethird oligonucleotide 300 nM of the template nucleic acid and 10 pg ofPCR template. A non-template control (NTC) was also tested. The reactionwas carried out in a total volume of 50 μl

The reaction was performed in a Mx3000P real-time PCR instrument underthe following parameters: 1 cycle at 95° C. for 2 minutes, followed by40 cycles of:

95° C. for 10 seconds

60° C. for 30 seconds

Fluorescence data was collected at the end of the 60° C. step of eachcycle.

The results are graphically represented in FIG. 18.

Example 9 Titration Curve of the Second Oligonucleotide

The following experiment was performed to determine the optimumconcentration of the second oligonucleotide in a method of theinvention.

Primers:

HPRT-Fwd: 5′-AATTATGGACAGGACTGAACGTCTTGCT-3′ HPRT-Rev:5′-TCCAGCAGGTCAGCAAAGAATTTATAGC-3′

Probes:

Second Oligonucleotide:

5′-BHQ2-CAGTTCAGGGGCCTCATCTCGGTTTTTCCCAGATGAGCCCCCAGAACTC-FAM-3′

The underlined portion of the second oligonucleotide corresponds to the5′ flap that was cleaved in the cleavage reaction. The flap wascomplementary to the first portion of the template nucleic acid andserved as a primer in subsequent extension reactions.

Third Oligonucleotide:

5′-tGaAcGaTgcc-Hex-3′

The third oligonucleotide was complementary to the second portion of thetemplate nucleic acid.

Template Nucleic Acid:

BHQ2-GGCATCGTTCAAAAGGAAAAACCGAGATGAGGCC-PO3

The template nucleic acid had a first region, which was complementary toand hybridizes with the cleaved flap (underlined), an extension regionand a second region, which was complementary to and hybridizes with thethird oligonucleotide (bolded). The template nucleic acid was blocked atthe 3′ nucleotide and was operably coupled to a quencher at the 5′nucleotide.

The oligonucleotides described above were used in a FullVelocity Q-PCRreaction (Stratagene, Calif.) according to the manufacturer'sinstructions. Briefly, the following reagents were combined in thereaction mixture: 1×FullVelocity buffer (containing dNTPs), Xul of theFullVelocity Multi-enzyme formulation, 400 nM the HPRT Fwd primer, 200nM of the HPRT Rev primer, 300 nM of the third oligonucleotide, 300 nMof the template nucleic acid and 100 fg of PCR template. The secondoligonucleotide was added to the reaction mixtures at variousconcentrations: 1000 nM, 200 n, 300 nM, 400 mM, 500 nM, 600 nM, 700 nMor 800 nM. A non-template control (NTC) was also tested. The reactionwas performed in 50 μl total reaction volume.

The reaction was performed in a Mx3000P real-time PCR instrument(Stratagene, Calif.) under the following reaction conditions: 1 cycle at95° C. for 2 minutes, followed by 50 cycles of:

95° C. for 30 seconds

60° C. for 60 seconds

Fluorescence data was collected at the end of the 60° C. step of eachcycle.

Fluorescence was detected for both the first cleavage reaction, HEX,(FIG. 19A) and the second cleavage reaction, FAM (FIG. 19B) at eachconcentration of the second oligonucleotide.

Example 10 Titration Curve of the Third Oligonucleotide

The following experiment was performed to determine the optimumconcentration of the third oligonucleotide in a method of the invention.

Primers:

HPRT-Fwd: 5′-AATTATGGACAGGACTGAACGTCTTGCT-3′ HPRT-Rev:5′-TCCAGCAGGTCAGCAAAGAATTTATAGC-3′

Probes:

Second Oligonucleotide:

5′--BHQ2- CAGTTCAGGGGCCTCATCTCGGTTTTTCCCAGATGAGCCCCCAGAACTC -FAM-3′

The underlined portion of the second oligonucleotide corresponds to the5′ flap that was cleaved in the cleavage reaction. The flap wascomplementary to the first portion of the template nucleic acid andserved as a primer in subsequent extension reactions.

Third Oligonucleotide:

tGaAcGaTgcc-Hex

The third oligonucleotide was complementary to the second portion of thetemplate nucleic acid.

Template Nucleic Acid:

5′-BHQ2-GGCATCGTTCAAAAGGAAAAACCGAGATGAGGCC-PO3-3′

The template nucleic acid had a first region, which was complementary toand hybridize with the cleaved flap (bolded), an extension region and asecond region, which was complementary to and hybridizes with the thirdoligonucleotide (underlined). The template nucleic acid was blocked atthe 3′ nucleotide and was attached to a quencher at the 5′ nucleotide.

The oligonucleotides described above were used in a FullVelocity Q-PCRreaction (Stratagene, Calif.) according to the manufacturer'sinstructions. Briefly, the following reagents were combined in thereaction mixture: 1×FullVelocity buffer (containing dNTPs), Xul of theFullVelocity Multi-enzyme formulation, 400 nM the HPRT Fwd primer, 200nM of the HPRT Rev primer, 500 nM of the second oligonucleotide, 300 nMof the template nucleic acid and 100 fg of PCR template. In order todetermine the optimal concentration of the third oligonucleotide thethird oligonucleotide was added to the reaction mixtures at varyingconcentrations: 100 nM, 200 n, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM,800 nM or 900 nM A non template control (NTC) was also tested. Thereaction was carried out in 50 μl total reaction volume.

The reaction was performed in a Mx3000P real-time PCR instrument(Stratagene, California) under the following reaction conditions: 1cycle at 95° C. for 2 minutes, followed by 50 cycles of:

95° C. for 10 seconds

60° C. for 30 seconds

Fluorescence data was collected at the end of the 60° C. step of eachcycle.

Fluorescence was determined for the first cleavage reaction, FAM, (FIG.20A) or the second cleavage reaction, HEX (FIG. 20B) for thirdoligonucleotide concentrations of 100 nM to 600 nM. Fluorescence wasalso determined for the first cleavage reaction, FAM, (FIG. 20C) or thesecond cleavage reaction, HEX for third oligonucleotide concentrationsof 700 nM to 900 nM.

1. A method of detecting a target nucleic acid, comprising (a)providing: said target nucleic acid, which comprises in 3′ to 5′ order afirst region, an extension region, and a second region, a templatenucleic acid, which comprises in 3′ to 5′ order a first region, anextension region, and a second region, a first oligonucleotide that isat least partially complementary to said first region of said targetnucleic acid, a second oligonucleotide comprising a 5′ region and a 3′region, wherein said 3′ region is at least partially complementary tosaid second region of said target nucleic acid and wherein said 5′region is not complementary to said target nucleic acid but is at leastpartially complementary to said first region of said template nucleicacid, a third oligonucleotide comprising a 5′ region and a 3′ region,wherein said 3′ region is at least partially complementary to saidsecond region of said template nucleic acid and said 5′ region is notcomplementary to said template nucleic acid, a cleavage means, and apolymerization activity; (b) mixing said target nucleic acid, saidtemplate nucleic acid, said first oligonucleotide, said secondoligonucleotide, said third oligonucleotide, said cleavage means andsaid polymerization activity under a series of reaction conditions,wherein said series of reaction conditions permit: formation of a duplexbetween said target nucleic acid and each of said first oligonucleotideand said 3′ region of said second oligonucleotide, wherein said 5′region of said second oligonucleotide is a flap, extension of said firstoligonucleotide by polymerization of a nucleic acid strand complementaryto a length of said extension region of said target nucleic acidsufficient to form a first cleavage structure, cleavage of said firstcleavage structure within said second oligonucleotide by said cleavagemeans, thereby permitting release of said flap of said secondoligonucleotide, formation of a second duplex with said released flap ofsaid second oligonucleotide, said template nucleic acid, and said thirdoligonucleotide, wherein said 5′ region of said third oligonucleotide isa second flap, extension of said released flap by polymerization of anucleic acid strand complementary to a length of said extension regionof said template nucleic acid sufficient to form a second cleavagestructure, and cleavage of said second cleavage structure within saidthird oligonucleotide by said cleavage means, thereby permittingcleavage and release of said third oligonucleotide; and (c) detection ofa signal generated by said release of said third oligonucleotide fromsaid template nucleic acid.
 2. A method of detecting a target nucleicacid, comprising (a) providing: said target nucleic acid, whichcomprises in 3′ to 5′ order a first region, an extension region, and asecond region, a template nucleic acid, which comprises in 3′ to 5′order a first region, an extension region, and a second region, a firstoligonucleotide that is at least partially complementary to said firstregion of said target nucleic acid, a second oligonucleotide comprisinga 5′ region and a 3′ region, wherein said 3′ region is at leastpartially complementary to said second region of said target nucleicacid and wherein said 5′ region is not complementary to said targetnucleic acid but is at least partially complementary to said firstregion of said template nucleic acid, a third oligonucleotide comprisinga 5′ region and a 3′ region, wherein said 3′ region is at leastpartially complementary to said second region of said template nucleicacid and said 5′ region is at least partially complementary to a regionthat is 3′ of said second region, a cleavage means, and a polymerizationactivity; (b) mixing said target nucleic acid, said template nucleicacid, said first oligonucleotide, said second oligonucleotide, saidthird oligonucleotide said cleavage means and said polymerizationactivity under a series of reaction conditions, wherein said series ofreaction conditions permit: formation of a duplex between said targetnucleic acid and each of said first oligonucleotide and said 3′ regionof said second oligonucleotide, wherein said 5′ region of said secondoligonucleotide is a flap, extension of said first oligonucleotide bypolymerization of a nucleic acid strand complementary to a length ofsaid extension region of said target nucleic acid sufficient to form afirst cleavage structure, cleavage of said first cleavage structurewithin said second oligonucleotide by said cleavage means, therebypermitting release of said flap of said second oligonucleotide,formation of a second duplex with said released flap of said secondoligonucleotide, said template nucleic acid, and said thirdoligonucleotide, extension of said released flap by polymerization of anucleic acid strand complementary to a length of said extension regionof said template nucleic acid sufficient to form a second cleavagestructure, cleavage of said second cleavage structure within said thirdoligonucleotide by said cleavage means, thereby permitting cleavage andrelease of said third oligonucleotide; and (c) detection of a signalgenerated by said release of said third oligonucleotide from saidtemplate nucleic acid.
 3. The method of claim 1 or 2, wherein saidpolymerase lacks 5′ to 3′ exonuclease activity.
 4. The method of claim 1or 2, wherein said polymerase is a DNA polymerase.
 5. The method ofclaim 1 or 2, wherein said polymerase is thermostable.
 6. The method ofclaim 1 or 2, wherein said polymerization comprises strand displacementactivity.
 7. The method of claim 1 or 2, wherein a single enzymecomprises said polymerization activity and said cleavage means.
 8. Themethod of claim 7, wherein said enzyme is E. coli DNA polymerase I, T7DNA polymerase, Tth DNA polymerase, or Taq DNA polymerase.
 9. The methodof claim 1 or 2, wherein a first enzyme comprises a polymerizationactivity and a second enzyme comprises said cleavage means.
 10. Themethod of claim 1 or 2, wherein said cleavage means comprises a 5′nuclease activity.
 11. The method of claim 1 or 2, wherein said cleavagemeans comprises a FEN-1 nuclease.
 12. The method of claim 11, whereinsaid FEN-1 nuclease is a flap specific nuclease.
 13. The method of claim1, wherein said FEN-1 nuclease is thermostable.
 14. The method of claim9, wherein said first and second enzymes are provided in a singleformulation.
 15. The method of claim 9, said first enzyme is a Pfu DNAPolymerase and said second enzyme is a FEN-1 nuclease.
 16. The method ofclaim 1 or 2, wherein said series of reaction conditions are nucleicacid polymerization reaction conditions.
 17. The method of claim 16,wherein said nucleic acid polymerization reaction conditions arepolymerase chain reaction conditions.
 18. The method of claim 1 or 2,wherein said extension reaction polymerizes nucleotides complementary toa length of the extension region sufficient to form a cleavagestructure, the polymerized complementary nucleic acid is adjacent at its3′ end to the downstream oligonucleotide in the duplex.
 19. The methodof claim 1 or 2, wherein said extension reaction polymerizes nucleotidescomplementary to a length of the extension region sufficient to form acleavage structure said length of said extension region is a length suchthat said extension region is a fully duplexed nucleic acid.
 20. Themethod of claim 1 or 2, wherein said extension reaction polymerizesnucleotides complementary to a length of the extension region sufficientto form a cleavage structure, said length of said extension region issuch that the template strand of said extension region comprises a 2nucleotide gap between the 3′ end of the complementary polymerizednucleic acid and the downstream oligonucleotide.
 21. The method of claim1 or 2, wherein said extension reaction polymerizes nucleotidescomplementary to a length of the extension region sufficient to form acleavage structure, said length of said extension region is such thatthe template strand of said extension region comprises a 4 nucleotidegap between the 3′ end of the complementary polymerized nucleic acid andthe downstream oligonucleotide.
 22. The method of claim 1 or 2, whereinsaid extension reaction polymerizes nucleotides complementary to alength of the extension region sufficient to form a cleavage structure,said length of said extension region is such that the template strand ofsaid extension region comprises a 6 nucleotide gap between the 3′ end ofthe complementary polymerized nucleic acid and the downstreamoligonucleotide.
 23. The method of claim 1 or 2, wherein said extensionreaction polymerizes nucleotides complementary to a length of theextension region sufficient to form a cleavage structure, said length ofsaid extension region is such that the template strand of said extensionregion comprises a 8 nucleotide gap between the 3′ end of thecomplementary polymerized nucleic acid and the downstreamoligonucleotide.
 24. The method of claim 1 or 2, wherein said extensionreaction polymerizes nucleotides complementary to a length of theextension region sufficient to form a cleavage structure, said length ofsaid extension region is such that the template strand of said extensionregion comprises a 10 nucleotide gap between the 3′ end of thecomplementary polymerized nucleic acid and the downstreamoligonucleotide.
 25. The method of claim 1 or 2, wherein said secondoligonucleotide does not comprise a detectable label.
 26. The method ofclaim 1 or 2, wherein said template nucleic acid further comprises afirst member of a pair of interactive labels and said thirdoligonucleotide further comprises a second member of said pair ofinteractive labels.
 27. The method of claim 26, wherein said firstmember of said pair of interactive labels is operatively coupled to the5′ nucleotide of said template nucleic acid.
 28. The method of claim 26,wherein said second member of said pair of interactive labels isoperatively coupled to the 3′ nucleotide of said third oligonucleotide.29. The method of claim 26, wherein said pair of interactive labelscomprises a fluorophore and a quencher.
 30. The method of claims 26,wherein said detection of a signal comprises detecting a change influorescent between said first and second members of said pair ofinteractive labels upon release of said third oligonucleotide.
 31. Themethod of claim 1 or 2, wherein said 5′ portion of said secondoligonucleotide is at least partially complementary to said 3′ portionof said second oligonucleotide.
 32. The method of claim 1 or 2, whereinsaid 3′ portion and said 5′ portion of said second oligonucleotide forma stem structure when the second oligonucleotide is not hybridize to thetarget nucleic acid.
 33. The method of claim 1 or 2, wherein said secondoligonucleotide further comprise a linker sequence between said 5′portion and said 3′ portion of said second oligonucleotide.
 34. Themethod of claim 33, wherein said linker portion is not complementary tosaid target nucleic acid but is complementary to the first portion ofsaid template nucleic acid.
 35. The method of claim 1 or 2, wherein the3′ nucleotide of said second oligonucleotide is blocked.
 36. The methodof claim 1 or 2, wherein the 3′ nucleotide of said template nucleic acidis blocked.
 37. The method of claim 1 or 2, wherein said polymerizationactivity is provided by a polymerase.
 38. The method of claim 1 or 2,wherein said first and said second cleavage structures are formed by thedisplacement of said second and said third oligonucleotides by saidpolymerase.
 39. The method of claim 1 or 2, wherein said series ofreaction conditions are performed on a solid substrate.
 40. Acomposition comprising: (i) a target nucleic acid, which comprises in 3′to 5′ order a first region, an extension region, and a second region;(ii) a template nucleic acid, which comprises in 3′ to 5′ order a firstregion, an extension region, and a second region and wherein saidtemplate nucleic acid has a first member of a pair of interactivelabels; (iii) a first oligonucleotide that is at least partiallycomplementary to said first region of said target nucleic acid; (iv) asecond oligonucleotide comprising a 5′ region and a 3′ region, whereinsaid 3′ region is at least partially complementary to said second regionof said target nucleic acid and wherein said 5′ region is notcomplementary to said target nucleic acid but is at least partiallycomplementary to said first region of said template nucleic acid; and(v) a third oligonucleotide comprising a 5′ region and a 3′ region,wherein said 3′ region is at least partially complementary to saidsecond region of said template nucleic acid and said 5′ region is notcomplementary to said template nucleic acid and wherein said thirdoligonucleotide has a second member of said pair of interactive labels.41. A composition comprising: (i) a target nucleic acid, which comprisesin 3′ to 5′ order a first region, an extension region, and a secondregion, (ii) a template nucleic acid, which comprises in 3′ to 5′ ordera first region, an extension region, and a second region and whereinsaid template nucleic acid has a first member of a pair of interactivelabels (iii) a first oligonucleotide that is at least partiallycomplementary to said first region of said target nucleic acid, and (iv)a second oligonucleotide comprising a 5′ region and a 3′ region, whereinsaid 3′ region is at least partially complementary to said second regionof said target nucleic acid and wherein said 5′ region is notcomplementary to said target nucleic acid but is at least partiallycomplementary to said first region of said template nucleic acid; and(v) a third oligonucleotide comprising a 5′ region and a 3′ region,wherein said 3′ region is at least partially complementary to the secondregion of the template nucleic acid and said 5′ region is at leastpartially complementary to a region that is 3′ of said second region ofsaid template nucleic acid and wherein said third oligonucleotide has asecond member of said pair of interactive labels.
 42. The composition ofclaim 40 or 41, further comprising a cleavage means.
 43. The compositionof claim 40 or 41, further comprising a polymerase.
 44. A kit comprisinga composition of claim 40 or 41, and packaging materials therefore. 45.The kit of claim 44, further comprising a cleavage means.
 46. The kit ofclaim 44, further comprising a nucleic acid polymerase.